Reaction vessel and temperature control system

ABSTRACT

A reaction vessel has a reaction chamber, a loading reservoir connected to the reaction chamber via a first channel, and an aspiration port connected to the chamber via a second channel. To load the sample into the reaction chamber, the sample is dispensed into the loading reservoir and then drawn into the chamber by application of a vacuum to the aspiration port. A system for controlling the temperature of the sample in the reaction vessel includes one or more thermal elements for heating or cooling the sample and optionally optics for detecting one or more analytes in the sample.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of commonly assigned, U.S.patent application Ser. No. 09/497,848, entitled “Reaction Vessel andTemperature Control System,” filed Feb. 4, 2000, now U.S. Pat. No.6,403,037, the entire disclosure of which is incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to a reaction vessel and temperature controlsystem.

BACKGROUND OF THE INVENTION

There are many applications in the field of chemical processing in whichit is desirable to precisely control the temperature of a sample, toinduce rapid temperature changes in the sample, and to detect targetanalytes in the sample. Applications for such heat-exchanging chemicalreactions may encompass organic, inorganic, biochemical or molecularreactions. Examples of thermal chemical reactions include isothermalnucleic acid amplification, thermal cycling nucleic acid amplification,such as the polymerase chain reaction (PCR), ligase chain reaction(LCR), self-sustained sequence replication, enzyme kinetic studies,homogeneous ligand binding assays, and more complex biochemicalmechanistic studies that require complex temperature changes.Temperature control systems also enable the study of certain physiologicprocesses where a constant and accurate temperature is required.

One of the most popular uses of temperature control systems is for theperformance of PCR to amplify a segment of nucleic acid. In this wellknown methodology, a DNA template is used with a thermostable DNApolymerase, nucleoside triphosphates, and two oligonucleotides withdifferent sequences, complementary to sequences that lie on oppositestrands of the template DNA and which flank the segment of DNA that isto be amplified (“primers”). The reaction components are cycled betweena higher temperature (e.g., 95° C.) for dehybridizing double strandedtemplate DNA, followed by lower temperatures (e.g., 40–60° C. forannealing of primers and 70–75° C. for polymerization). Repeated cyclingbetween dehybridization, annealing, and polymerization temperaturesprovides exponential amplification of the template DNA.

Nucleic acid amplification may be applied to the diagnosis of geneticdisorders; the detection of nucleic acid sequences of pathogenicorganisms in a variety of samples including blood, tissue,environmental, air borne, and the like; the genetic identification of avariety of samples including forensic, agricultural, veterinarian, andthe like; the analysis of mutations in activated oncogenes, detection ofcontaminants in samples such as food; and in many other aspects ofmolecular biology. Polynucleotide amplification assays can be used in awide range of applications such as the generation of specific sequencesof cloned double-stranded DNA for use as probes, the generation ofprobes specific for uncloned genes by selective amplification ofparticular segments of cDNA, the generation of libraries of cDNA fromsmall amounts of mRNA, the generation of large amounts of DNA forsequencing and the analysis of mutations.

A preferred detection technique for chemical or biochemical analysis isoptical interrogation, typically using fluorescence or chemiluminescencemeasurements. For ligand-binding assays, time-resolved fluorescence,fluorescence polarization, or optical absorption is often used. For PCRassays, fluorescence chemistries are often employed.

Some conventional instruments for conducting thermal reactions and foroptically detecting the reaction products incorporate a block of metalhaving as many as ninety-six conical reaction tubes. The metal block isheated and cooled either by a Peltier heating/cooling apparatus or by aclosed-loop liquid heating/cooling system in which liquid flows throughchannels machined into the block. Such instruments incorporating a metalblock are described in U.S. Pat. No. 5,038,852 to Johnson and U.S. Pat.No. 5,333,675 to Mullis.

These conventional instruments have several disadvantages. First, due tothe large thermal mass of a metal block, the heating and cooling ratesin these instruments are limited, resulting in longer processing times.For example, in a typical PCR application, fifty cycles may require twoor more hours to complete. With these relatively slow heating andcooling rates, some processes requiring precise temperature control areinefficient. For example, reactions may occur at the intermediatetemperatures, creating unwanted and interfering side products, such asPCR “primer-dimers” or anomalous amplicons, which are detrimental to theanalytical process. Poor control of temperature also results inover-consumption of expensive reagents necessary for the intendedreaction.

Some of the instrumentation for newer processes requiring faster thermalcycling times has recently become available. One such device isdisclosed by Northrup et al. in U.S. Pat. No. 5,589,136. The deviceincludes a silicon-based, sleeve-type reaction chamber that combinesheaters, such as doped polysilicon for heating, and bulk silicon forconvection cooling. The device optionally includes a secondary tube(e.g., plastic) for holding the sample. In operation, the tubecontaining the sample is inserted into the silicon sleeve. Each sleevealso has its own associated optical excitation source and fluorescencedetector for obtaining real-time optical data.

A different thermal cycling instrument is available from IdahoTechnologies. This instrument employs forced-air heating and cooling ofcapillary sample carriers mounted in a carousel. The instrument monitorseach capillary sample carrier in sequence as the capillary samplecarriers are rotated past an optical detection site.

SUMMARY

The present invention provides an improved instrument and reactionvessel for thermally controlling and optically interrogating a sample.In preferred aspects, the system of the present invention permitsconvenient and effective loading of a sample into the reaction vessel,rapid heating and cooling of the sample, optimal thermal transferbetween the sample and heating or cooling elements, and real-timeoptical detection and monitoring of the sample with increased detectionsensitivity.

In accordance with an aspect of the present invention, a system forcontrolling the temperature of a sample comprises a reaction vesselhaving a reaction chamber defined by two opposing major walls and sidewalls connecting the major walls to each other. At least one of themajor walls comprises a sheet or film. The vessel also has a loadingreservoir for receiving the sample prior to loading the sample into thereaction chamber. The loading reservoir is connected to the reactionchamber via a first channel. The vessel further has an aspiration portconnected to the reaction chamber via a second channel. The system alsoincludes an aspiration device for establishing a seal with theaspiration port and for drawing the sample from the loading reservoirinto the reaction chamber. The vessel further includes flow controlmeans for preventing substantial flow of the sample from the loadingreservoir into the reaction chamber until the sample is drawn into thechamber by the aspiration device. The system further includes at leastone heating or cooling surface for contacting the sheet or film, thesheet or film being sufficiently flexible to conform to the surface. Atleast one thermal element heats or cools the surface to induce atemperature change in the reaction chamber.

In some embodiments, the flow control means comprises at least oneportion of the first channel having a sufficiently small width ordiameter to prevent substantial flow of the sample from the loadingreservoir to the chamber until the sample is drawn into the chamber bythe aspiration device. In some embodiments, the flow control meanscomprises at least one valve, membrane, or screen in the first channel.

In a specific embodiment, at least two of the side walls of the reactionchamber are optically transmissive, and the system further includesoptics for optically interrogating the reaction chamber while the sheetor film is in contact with the heating or cooling surface. The opticscomprise at least one light source for transmitting light to the chamberthrough a first one of the optically transmissive walls and at least onedetector for detecting light exiting the chamber through a second one ofthe optically transmissive walls. The system also preferably comprisesat least one controller for controlling the operation of the thermalelement, light source, and detector.

In a specific embodiment, the system further comprises means forincreasing the pressure in the reaction chamber. The pressure increasein the reaction chamber is sufficient to force the sheet or film toconform to the heating or cooling surface, ensuring optimal heattransfer to or from the reaction chamber. In some embodiments, thevessel further includes a seal aperture extending over an outer end ofthe loading reservoir and an outer end of the aspiration port, and themeans for increasing the pressure in the chamber comprises a plug thatis insertable into the aperture to compress gas in the vessel.

In accordance with another aspect of the invention, a system forcontrolling the temperature of a sample comprises a reaction vesselhaving a reaction chamber and having a loading reservoir for receivingthe sample prior to loading the sample into the reaction chamber. Theloading reservoir is connected to the reaction chamber via a firstchannel. The vessel also has an aspiration port connected to thereaction chamber via a second channel. The vessel further has a sealaperture extending over an outer end of the loading reservoir and anouter end of the aspiration port. The system also includes an aspirationdevice for establishing a seal with the aspiration port and for drawingthe sample from the loading reservoir into the reaction chamber. Thevessel includes flow control means for preventing substantial flow ofthe sample from the loading reservoir into the reaction chamber untilthe sample is drawn into the reaction chamber by the aspiration device.The system further includes a plug that is insertable into the aperture,after loading the sample into the reaction chamber, to simultaneouslyseal the loading reservoir, reaction chamber, and aspiration port fromthe external environment. At least one thermal element heats or coolsthe reaction chamber.

In a specific embodiment, the reaction chamber is defined by a pluralityof walls, at least two of the walls being optically transmissive toprovide optical windows to the reaction chamber, and the system furtherincludes optics for optically interrogating the reaction chamber. Theoptics comprise at least one light source for transmitting light to thechamber through a first one of the optically transmissive walls and atleast one detector for detecting light exiting the chamber through asecond one of the optically transmissive walls. The system alsopreferably comprises at least one controller for controlling theoperation of the thermal element, light source, and detector.

In accordance with another aspect of the invention, there is provided asystem for loading a sample into a reaction vessel and for controllingthe temperature of the sample in the vessel. The vessel includes areaction chamber, a loading reservoir connected to the reaction chambervia a first channel, an aspiration port connected to the reactionchamber via a second channel, and a seal aperture extending over anouter end of the loading reservoir and an outer end of the aspirationport. The system comprises an aspiration and dispensing device fordispensing the sample into the loading reservoir, for establishing aseal with the aspiration port, and for drawing the sample from theloading reservoir into the reaction chamber. The vessel includes flowcontrol means for preventing substantial flow of the sample from theloading reservoir into the reaction chamber until the sample is drawninto the reaction chamber by the aspiration and dispensing device. Thesystem also comprises an automated machine for inserting a plug into theseal aperture after loading the sample into the chamber. The systemfurther comprises one or more thermal elements for heating or coolingthe reaction chamber.

In some embodiments, the reaction chamber is defined by a plurality ofwalls, at least two of the walls being optically transmissive, and thesystem further comprises optics for optically interrogating the chamber.The optics comprise at least one light source for transmitting light tothe chamber through a first one of the optically transmissive walls andat least one detector for detecting light exiting the chamber through asecond one of the optically transmissive walls. The system alsopreferably includes at least one controller for controlling theoperation of the thermal element, light source, and detector.

In accordance with another aspect of the invention, a reaction vesselcomprises a reaction chamber defined by two opposing major walls andside walls connecting the major walls to each other. At least one of themajor walls comprises a sheet or film sufficiently flexible to conformto a heating or cooling surface. At least two of the side walls areoptically transmissive to provide optical windows to the reactionchamber. The vessel also has a loading reservoir for receiving thesample prior to loading the sample into the reaction chamber. Theloading reservoir is connected to the reaction chamber via a firstchannel. The vessel further includes an aspiration port for establishinga seal with an aspiration device. The aspiration port is connected tothe reaction chamber via a second channel, thereby enabling theaspiration device to draw the sample from the loading reservoir into thechamber. The vessel also includes flow control means for preventingsubstantial flow of the sample from the loading reservoir into thereaction chamber until the sample is drawn into the reaction chamber bythe aspiration device.

In some embodiments, the flow control means comprises at least oneportion of the first channel having a sufficiently small width ordiameter to prevent substantial flow of the sample from the loadingreservoir to the chamber until the sample is drawn into the chamber bythe aspiration device. In some embodiments, the flow control meanscomprises at least one valve, membrane, or screen in the first channel.

In accordance with another aspect of the invention, a reaction vesselcomprises a reaction chamber and a loading reservoir for receiving thesample prior to loading the sample into the reaction chamber. Theloading reservoir is connected to the reaction chamber via a firstchannel. The vessel also has an aspiration port for establishing a sealwith an aspiration device, the aspiration port being connected to thereaction chamber via a second channel thereby enabling the aspirationdevice to draw the sample from the loading reservoir into the reactionchamber. The vessel further includes flow control means for preventingsubstantial flow of the sample from the loading reservoir into thereaction chamber until the sample is drawn into the reaction chamber bythe aspiration device. The vessel also comprises first and second plugsfor sealing the loading reservoir and the aspiration port, respectively,and for compressing gas in the vessel, thereby increasing pressure inthe reaction chamber.

In accordance with another aspect of the invention, a reaction vesselcomprises a reaction chamber and a loading reservoir for receiving thesample prior to loading the sample into the reaction chamber. Theloading reservoir is connected to the reaction chamber via a firstchannel. The vessel also has an aspiration port for establishing a sealwith an aspiration device, the aspiration port being connected to thereaction chamber via a second channel, thereby enabling the aspirationdevice to draw the sample from the loading reservoir into the reactionchamber. The vessel further includes flow control means for preventingsubstantial flow of the sample from the loading reservoir into thereaction chamber until the sample is drawn into the reaction chamber bythe aspiration device. The vessel also has a seal aperture, extendingover an outer end of the loading reservoir and an outer end of theaspiration port, for receiving a plug that is inserted into the apertureafter loading the sample into the reaction chamber.

In some embodiments, the system of the present invention is configuredas a small hand-held instrument, and in other embodiments, as a largeinstrument with multiple reaction sites for simultaneously processinghundreds of samples. In high throughput embodiments, the heating and/orcooling elements and optics are preferably disposed in a single housingto form an independently controllable, heat-exchanging module withdetection capability. The system includes a base instrument forreceiving a plurality of such modules, and the base instrument includesprocessing electronics for independently controlling the operation ofeach module. Each module provides a reaction site for thermallyprocessing a sample contained in a reaction vessel and for detecting oneor more target analytes (e.g., nucleic acid sequences of interest) inthe sample. The system may also include a computer for controlling thebase instrument.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a partially exploded, isometric view of a reaction vesselhaving a reaction chamber. The major walls of the reaction chamber areremoved, for illustrative purposes, to show the interior of the chamber.

FIG. 2A is a schematic, front view of the vessel of FIG. 1.

FIG. 2B is a schematic, front view of a reaction vessel having a valve.

FIG. 2C is a schematic, front view of a reaction vessel having amembrane or screen.

FIG. 3 is another front view of the vessel of FIG. 1.

FIG. 4 is a side view of the vessel of FIG. 1 inserted into a thermalsleeve formed by opposing plates.

FIG. 5 is a front view of one of the plates of FIG. 4.

FIG. 6 is a schematic, front view of a heat-exchanging module accordingto the present invention having a thermal sleeve, a pair of opticsassemblies, and a cooling system. The reaction vessel of FIG. 1 isinserted into the thermal sleeve.

FIG. 7 is an exploded view of a support structure for holding the platesof FIG. 4.

FIGS. 8–9 are assembled views of the support structure of FIG. 7.

FIG. 10 is an isometric view showing the exterior of one the opticsassemblies of FIG. 6.

FIG. 11 is an isometric view of the plates of FIG. 4 in contact with theoptics assembly of FIG. 10 with the vessel of FIG. 1 positioned abovethe plates.

FIG. 12 is an isometric view of the vessel of FIG. 1 inserted betweenthe plates of FIG. 4.

FIGS. 13A and 13B are graphs showing the excitation and emissionspectra, respectively, of four dyes often used in thermal reactions.

FIG. 13C shows the effects of filtering the outputs of green and blueLEDs to provide distinct excitation wavelength ranges.

FIG. 13D shows the effects of filtering light emitted from each of thefour dyes of FIGS. 13A–B to form distinct emission wavelength ranges.

FIG. 14 is a plan view of an optical excitation assembly of the moduleof FIG. 6.

FIG. 15 is an exploded view of the excitation assembly of FIG. 14.

FIG. 16 is a plan view of an optical detection assembly of the module ofFIG. 6.

FIG. 17 is an exploded view of the detection assembly of FIG. 16.

FIG. 18 is an isometric view of a multi-site reactor system according toan embodiment present invention.

FIG. 19 is a schematic, block diagram of another multi-site reactorsystem having multiple thermal cycling instruments daisy-chained to acomputer and a power source.

FIG. 20 is a schematic, block diagram of a base instrument of the systemof FIG. 18.

FIG. 21 is a schematic, block diagram of the electronic components ofthe module of FIG. 6.

FIG. 22 is a schematic diagram of a pick-and-place machine having apipette for filling the vessel of FIG. 1.

FIGS. 23A–23D are schematic diagrams of the pick-and-place machine ofFIG. 22 filling and pressurizing a reaction vessel.

FIGS. 24A–24B are schematic, cross-sectional views of a plug beinginserted into an aperture of the vessel of FIG. 1.

FIG. 25A is a schematic, front view of a reaction vessel according toanother embodiment of the invention.

FIG. 25B is a schematic, front view of a reaction vessel having a valveaccording to another embodiment of the invention.

FIG. 25C is a schematic, front view of a reaction vessel having amembrane or screen according to another embodiment of the invention.

FIG. 26 is a schematic diagram of a pick-and-place machine forpressurizing a reaction vessel according to another embodiment of theinvention.

FIG. 27 is a schematic diagram of a pick-and-place machine using aneedle to pressurize a reaction vessel according to an alternativeembodiment of the invention.

FIGS. 28–29 are schematic diagrams of a press machine having a platenfor sealing a port of a reaction vessel according to another embodimentof the invention.

FIG. 30 is a schematic, cross sectional view of a reaction vesselaccording to another embodiment of the invention.

DETAILED DESCRIPTION

The present invention provides a reaction vessel and system forthermally controlling and optically interrogating a sample. The samplemay be a solution or suspension containing particles, cells,microorganisms, ions, or small and large molecules, such as proteins andnucleic acids, etc. In a particular use, the sample may be a bodilyfluid (e.g., blood, urine, saliva, sputum, seminal fluid, spinal fluid,mucus, or other bodily fluids). Alternatively, the sample may be a solidmade soluble or suspended in a liquid or the sample may be anenvironmental sample such as ground or waste water, soil extracts, orpesticide residues. Further, the sample may be mixed with one or morechemicals, reagents, diluents, or buffers. The term “sample” isunderstood to encompass original samples of interest (e.g., bodilyfluids), samples containing at least parts of original samples, andreaction products resulting from reactions of original samples.

In a preferred embodiment, the system includes a reaction vessel forholding the sample and a heat-exchanging module into which the vessel isinserted for thermal processing and optical detection. Theheat-exchanging module includes a pair of opposing plates between whichthe vessel is inserted for thermal processing, one or more heating orcooling elements for heating or cooling the plates, and optics foroptically interrogating the sample contained in the vessel. The systemalso includes a base unit with processing electronics for receiving aplurality of such heat-exchanging modules and for independentlycontrolling each module. The system may also include a controller, suchas a personal computer or network computer, that provides a userinterface to the system and controls the operation of the base unit. Thesystem is useful for performing heat-exchanging chemical reactions, suchas nucleic acid amplification, and for detecting target analytes.

FIGS. 1–23D illustrate a preferred embodiment. FIG. 1 shows a partiallyexploded view of a reaction vessel 12 according to the preferredembodiment, and FIG. 2A shows a front view of the vessel 12. The vessel12 has a body defining a reaction chamber 17. The chamber 17 holds asample for thermal processing and optical interrogation. The reactionvessel 12 includes a rigid frame 16 that defines the side walls 19A,19B, 20A, 20B of the chamber 17. The vessel also includes thin, flexiblesheets or films attached to opposite sides of the rigid frame 16 to formtwo opposing major walls 18 of the chamber. (The major walls 18 areshown in FIG. 1 exploded from the rigid frame 16 for illustrativepurposes). The reaction chamber 17 is thus defined by the two opposingmajor walls 18, attached to opposite sides of the frame 16, and therigid side walls 19A, 19B, 20A, 20B that connect the major walls 18 toeach other.

The major walls 18 facilitate optimal thermal conductance to the samplecontained in the chamber 17. Each of the walls 18 is sufficientlyflexible to contact and conform to a respective heating or coolingsurface, thus providing for optimal thermal contact and heat transferbetween the heating or cooling surface and the sample contained in thechamber 17. Furthermore, the flexible walls 18 continue to conform tothe surfaces if the shape of the surfaces changes due to thermalexpansion or contraction during the course of the heat-exchangingoperation.

As shown in FIG. 4, the heating or cooling surfaces for contacting theflexible walls 18 are preferably formed by a pair of opposing plates50A, 50B positioned to receive the chamber 17 between them. When thechamber 17 of the vessel is inserted between the plates 50A, 50B, theinner surfaces of the plates contact the walls 18 and the flexible wallsconform to the surfaces of the plates. The plates are preferably spaceda distance from each other equal to the thickness T of the chamber 17 asdefined by the thickness of the frame 16. In this position, minimal orno gaps are found between the plate surfaces and the walls 18. Theplates may be heated and cooled by various thermal elements to inducetemperature changes within the chamber 17, as is described in greaterdetail below.

The walls 18 are preferably flexible films of polymeric material such aspolypropylene, polyethylene, polyester, or other polymers. The films mayeither be layered, e.g., laminates, or the films may be homogeneous.Layered films are preferred because they generally have better strengthand structural integrity than homogeneous films. In particular, layeredpolypropylene films are presently preferred because polypropylene is notinhibitory to PCR. Alternatively, the walls 18 may comprise any othermaterial that may be formed into a thin, flexible sheet and that permitsrapid heat transfer. For good thermal conductance, the thickness of eachwall 18 is preferably between about 0.003 to 0.5 mm, more preferablybetween 0.01 to 0.15 mm, and most preferably between 0.025 to 0.08 mm.

Referring again to FIGS. 1–2A, the reaction vessel 12 also includes aloading structure 30 extending from the frame 16 for loading the sampleinto the chamber 17. The loading structure 30 is preferably integrallyformed (e.g., molded in one piece) with the frame 16. The loadingstructure 30 defines a loading reservoir 38 in fluid communication withthe chamber 17. The loading reservoir 38 receives the sample prior toloading the sample into the chamber 17. The loading reservoir preferablyhas a volume capacity equal to or greater than the volume capacity ofthe chamber 17. The loading reservoir 38 is connected to the chamber 17by an inlet channel 39. The loading structure 30 also defines anaspiration port 41 connected to the chamber 17 by an outlet channel 43.The aspiration port 41 has tapered walls 45 for establishing a seal withan aspiration and dispensing device (e.g., a pipette), thereby enablingthe device to draw the sample from the loading reservoir 38, through thechannel 39, and into the chamber 17. In general, the aspiration anddispensing device may comprise a pipette, syringe, probe, luer fitting,or any other device capable of aspirating and dispensing a fluid.

The reaction vessel preferably includes flow control means forpreventing substantial flow of the sample from the loading reservoir 38to the reaction chamber 17 until the sample is drawn into the chamber 17by the aspiration device. As used herein, the phrase “substantial flowof the sample” is intended to mean flow of at least 50% of the sample.The flow control means should be sufficient to prevent at least 50% ofthe sample from flowing from the loading reservoir 38 into the chamber17 for the time period beginning when the sample is dispensed into theloading reservoir 38 and ending when the sample is drawn into thechamber 17 by the aspiration device (typically a time period in therange of 3 to 60 seconds). In most applications, the sample is drawninto the chamber 17 only a few seconds after loading the sample into theloading reservoir 38. In other applications, however, the sample may beheld in the loading reservoir 38 for longer periods of time whileadditional liquids or materials are added to the loading reservoir 38(e.g., reagents, probes, additional sample material). The additionalliquids or materials are mixed with the sample in the loading reservoir38, and then the mixture is drawn into the chamber 17.

Suitable flow control means include, but are not limited to: a channelhaving a narrow width or diameter; one or more valves; one or moremembranes; or one or more screens. FIG. 2A shows one embodiment of thevessel in which the flow control means is at least one portion of theinlet channel 39 having a sufficiently small width or diameter (e.g.,0.031 inches or less is suitable for most aqueous solutions having theviscosity of water) to prevent substantial flow of the sample (e.g., dueto gravitational force) from the loading reservoir 38 to the chamber 17until the sample is drawn into the chamber by the aspiration device.FIG. 2B shows another embodiment of the vessel in which the flow controlmeans is a valve 11 in the channel 39. There are many types of valvesknown in the art that are suitable for this purpose including, e.g., aflap valve, check valve, or rotary valve. FIG. 2C shows anotherembodiment of the vessel in which the flow control means is a membraneor screen 13 in the channel 39. Suitable membranes include, e.g.,burstable or piercable membranes that are burst or pierced to permitfluid flow through the channel 39. If a screen is used as the flowcontrol component, the screen should have a pore size sufficient toprevent substantial flow of the sample before a vacuum is applied to theaspiration port 41 and permit flow of the sample when the vacuum isapplied.

The flow control permits simple and accurate metering of the volume ofsample loaded into the chamber 17. For example, accurate loading of thesample into the chamber 17 may be performed as follows. A volume ofsample equal to the volume capacity of the chamber 17 (e.g., 100microliters) is initially placed in the loading reservoir 38 using apipette. The pipette is then inserted into the aspiration port 41 andused to suck the same amount of air (e.g., 100 microliters) out of thechamber 17, so that the correct volume of sample is sucked from theloading reservoir 38 into the chamber 17 to precisely fill the chamber.Alternatively, the volume of sample loaded into the chamber 17 may bemonitored visually, optically, or electronically.

Referring again to FIGS. 1–2A, the loading structure 30 also defines aseal aperture 14 that extends over the outer end of the loadingreservoir 38 and over the outer end of the aspiration port 41. Thevessel 12 includes a plug 22 that is inserted into the aperture 14 afterfilling the chamber 17 with the sample. The plug 22 seals the aperture14, and thus simultaneously seals the chamber 17, loading reservoir 38,and aspiration port 41 from the environment external to the vessel 12.The plug 22 also compresses gas in the vessel 12, thereby increasingpressure in the chamber 17 and outwardly expanding the flexible walls18. The gas compressed by the plug 22 is typically air filling theaperture 14, loading reservoir 38, and aspiration port 41. Thepressurization of the chamber 17 is important because the pressureincrease in the chamber forces the walls 18 against the surfaces of theplates 50A, 50B (see FIG. 4) and ensures that the walls 18 fully contactand conform to the inner surfaces of the plates, thus guaranteeingoptimal thermal conductance between the plates 50A, 50B and the chamber17.

The plug may comprise any device capable of establishing a seal with thewalls of the aperture 14 and of compressing gas in the vessel 12. Suchdevices include, but are not limited to, pistons, plungers, or stoppers.The plug 22 of the preferred embodiment includes a sealing ring 32 and astabilizer ring 34. When the plug 22 is inserted into the aperture 14,the sealing ring 32 establishes an annular seal with the inner walls 37of the aperture 14 and compresses air in the aperture. The stabilizerring 34 maintains the plug 22 in coaxial alignment with the aperture 14as the plug is inserted into the aperture. The plug 22 also includes acap 36 having latches 33. The vessel 12 includes corresponding catches35 extending from the sides of the loading structure 30. When the plug22 is inserted into the aperture 14, the catches 35 engage the latches33, thereby securing the plug in the aperture. As shown in FIG. 1, theplug 22 and aperture 14 are preferably oval in shape to give the vessel12 a slim profile. The slim profile enables multiple vessels to bespaced closely to each other (e.g., standard 9 mm spacing betweenvessels). It is to be understood, however, that the plug 22 and aperture14 may have any shape desired, e.g., circular, square, rectangular, etc.

The loading structure 30 has an inner surface 49 defining the aperture14. The inner surface 49 may optionally have one or more pressurecontrol grooves 42 formed therein. In the preferred embodiment, theinner surface has four pressure control grooves (only two visible in theisometric view of FIG. 1) spaced equidistantly about the circumferenceof the aperture 14. Referring now to FIG. 24A, the pressure controlgrooves 42 extend to a predetermined depth D₁ in the aperture 14. Thegrooves 42 allow gas to escape from the aperture 14 and thus preventpressurization of the chamber 17 until the sealing ring 32 reaches thedepth D₁ in the aperture. When the sealing ring 32 reaches the depth D₁,the sealing ring establishes an annular seal with the surface 49 andbegins to compress air trapped in the aperture 14, loading reservoir 38,and aspiration port 41. The compression of the trapped air causes thedesired pressurization of the chamber 17.

The insertion of the plug 22 into the aperture 14 is illustrated inFIGS. 24A–24B. As shown in FIG. 24A, prior to inserting the plug 22 intothe aperture 14, the chamber 17 is filled with a reaction mixture R,typically the sample mixed with one or more reagents. Specific methodsfor filling the chamber 17 are discussed in detail below. The reactionmixture R fills the vessel 12 to a liquid surface level S. Also prior toinserting the plug 22 into the aperture 14, the aperture 14 contains airhaving pressure equal to the pressure of the atmosphere external to thevessel, hereinafter called ambient pressure. The ambient pressure isusually standard atmospheric pressure, e.g., about 14.7 pounds persquare inch (psi). When the plug 22 is first inserted into the aperture14, the sealing ring 32 begins to displace the air in the aperture. Thedisplaced air escapes from the aperture 14 through the pressure controlgrooves 42.

Referring now to FIG. 24B, when the sealing ring 32 reaches the depth D₁at which the pressure control grooves end, the sealing ring 32establishes an annular seal with the walls of the aperture 14 and beginsto compress air trapped in the vessel between the sealing ring 32 andthe surface level S of the reaction mixture. The reaction mixture isusually a liquid and therefore substantially incompressible by the plug.The air trapped in the vessel 12, however, may be compressed to increasepressure in the chamber 17. As the plug 22 is inserted further into theaperture 14, the ring 34 keeps the plug 22 coaxially aligned with theaperture 14 as the sealing ring 32 continues to compress air.

When the plug 22 is fully inserted, the sealing ring 32 seals theaperture 14 at a depth D₂ which is lower than the depth D₁ at which thepressure control grooves 42 terminate. The distance D₃ traveled by thesealing ring 32 between depths D₁ and D₂, i.e. the distance of thepressure stroke, determines the amount of pressurization of the chamber17. Referring again to FIG. 4, the pressure in the chamber 17 should besufficiently high to ensure that the flexible major walls 18 of thechamber outwardly expand to contact and conform to the inner surfaces ofthe plates 50A, 50B. The pressure should not be so great, however, thatthe flexible walls 18 burst, become unattached from the rigid frame 16,or deform the frame or plates.

It is presently preferred to pressurize the chamber 17 to a pressure inthe range of 2 to 50 psi above ambient pressure. This range is presentlypreferred because 2 psi is generally enough pressure to ensureconformity between the flexible walls 18 and the surfaces of the plates50A, 50B, while pressures above 50 psi may cause bursting of the walls18 or deformation of the frame 16 or plates 50A, 50B. More preferably,the chamber 17 is pressurized to a pressure in the range of 8 to 15 psiabove ambient pressure. This range is more preferred because it issafely within the practical limits described above, i.e. pressures of 8to 15 psi are usually more than enough to ensure that the flexible walls18 contact and conform to the surfaces of the plates 50A, 50B, but aresignificantly lower than the pressures that might burst the walls 18 ordeform the frame 16.

Referring again to FIG. 24B, the desired pressurization of the chamber17 may be achieved by proper design of the plug 22, aperture 14, andpressure control grooves 42 and by use of the equation:P ₁ *V ₁ =P ₂ *V ₂;

where:

P₁ is equal to the pressure in the vessel 12 prior to insertion of theplug 22;

V₁ is equal to the volume capacity of the vessel between the liquidsurface level S and the depth D₁ to which the pressure control grooves42 extend;

P₂ is equal to the desired final pressure in the chamber 17 afterinsertion of the plug 22 into the aperture 14; and

V₂ is equal to the volume capacity of the vessel between the liquidsurface level S and the depth D₂ at which the sealing ring 32establishes a seal with the walls of the aperture 14 when the plug 22 isfully inserted into the vessel.

To ensure the desired pressurization P₂ of the chamber 17, one shouldsize the aperture 14 and pressure stroke distance D₃ such that the ratioof the volumes V₁:V₂ is equal to the ratio of the pressures P₂:P₁. Anengineer having ordinary skill in the art will be able to selectsuitable values for the volumes V₁ and V₂ using the description andequation given above. For example, in the presently preferredembodiment, the initial pressure P₁ in the vessel is equal to standardatmospheric pressure of about 14.7 psi, the volume V₁ is equal to 500 l,the depth D₁ is equal to 0.2 inches, the depth D₂ is equal to 0.34inches to give a pressure stroke distance D₃ of 0.14 inches, and thevolume V₂ is equal to 275 l to give a final pressure P₂ of about 26.7psi (the desired 12 psi above ambient pressure). This is just oneexample of suitable dimensions for the vessel 12 and is not intended tolimit the scope of the invention. Many other suitable values may beselected.

In selecting suitable dimensions for the aperture 14 and pressure strokedistance D₃ (and thus the volumes V₁, V₂), there is no theoretical limitto how large or small the dimensions may be. It is only important thatthe ratio of the volumes V₁:V₂ yield the desired final desired pressureP₂ in the chamber. As a practical matter, however, it is presentlypreferred to design the vessel such that the distance D₃ of the pressurestroke is at least 0.05 inches, i.e., so that the plug 22 when fullyinserted into the aperture 14 extends to a depth D₂ that is at least0.05 inches below the depth D₁ at which the pressure control groovesend. This minimum length of the pressure stroke is preferred to reduceor make negligible the effect that any manufacturing or operating errorsmay have on the pressurization of the chamber. For example, the lengthof the pressure stroke may differ slightly from vessel to vessel due tomanufacturing deviations, or the volume of air compressed may vary dueto operator error in filling the vessel (e.g., different fill levels).If the vessel is designed to have a sufficiently long pressure stroke,however, such variances will have a lesser or negligible effect on theratio of volumes V_(i):V₂ and suitable pressurization of the chamberwill still occur.

In addition, to provide a safety margin for manufacturing or operatorerrors, one should select a pressure stroke sufficient to achieve afinal pressure P₂ that is safely higher (e.g., at least 3 psi higher)than the minimum pressure needed to force the flexible walls of thechamber against the inner surfaces of the plates. With such a safetymargin, any deviations in the final pressure due to manufacturingdeviations or errors in filling the chamber will have a negligibleeffect and suitable pressurization of the chamber 17 will still occur.As stated above, the plunger stroke is preferably designed to increasepressure in the chamber 17 to a pressure in the range of 8 to 15 psiabove ambient pressure to provide the safety margin.

The pressure control grooves 42 provide several advantages. First, thepressure control grooves 42 provide a simple mechanism for precisely andaccurately controlling the pressure stroke of the plug 22, and hence thepressurization of the chamber 17. Second, the pressure control grooves42 allow the plug 22 to become fully aligned with the aperture 14 beforethe pressure stroke begins and thus prevent the plunger from becomingmisaligned or cocked in the channel. This ensures a highly consistentpressure stroke. Although it is possible for the vessel to have only onepressure control groove, it is preferable for the vessel to havemultiple pressure control grooves (e.g., 2 to 6 grooves) spacedequidistantly about the circumference of the aperture 14. Referringagain to FIG. 24A, the pressure control grooves 42 preferably cut about0.01 to 0.03 inches into the surface 49 defining the aperture 14. Thisrange is preferred so that the pressure control grooves 42 are largeenough to allow air to escape from the aperture 14, but do not cut sodeeply into the surface 41 that they degrade the structural integrity ofthe loading structure 30.

Although the pressure control grooves 42 are advantageous, they are nota mandatory feature of the vessel 12. It is possible to construct thevessel 12 without the pressure control grooves and still achieve thedesired pressurization of the chamber 17. Embodiments in which thevessel lacks pressure control grooves are intended to fall within thescope of the present invention. In embodiments in which the vessel lackspressure control grooves, the pressure stroke of the plug 22 begins whenthe sealing ring 32 enters the aperture 14 and establishes a seal withthe walls of the aperture. In these embodiments, the volume V₁ (for usein the equation above) is equal to the volume capacity of the vessel 12between the liquid surface level S and the top of the loading structure30 where the sealing ring 32 first establishes a seal with the innersurface 49.

Referring again to FIG. 2A, the vessel 12 also preferably includesoptical windows for in situ optical interrogation of the sample in thechamber 17. In the preferred embodiment, the optical windows are theside walls 19A, 19B of the rigid frame 16. The side walls 19A, 19B areoptically transmissive to permit the transmission of light to thechamber 17 through the side wall 19A and detection of light exiting thechamber 17 through the side wall 19B. Arrows A represent illuminationbeams entering the chamber 17 through the side wall 19A and arrows Brepresent emitted light (e.g., fluorescent emission from labeledanalytes in the sample) exiting the chamber 17 through the side wall19B.

The side walls 19A, 19B are preferably angularly offset from each other.It is usually preferred that the walls 19A, 19B are offset from eachother by an angle of about 90°. A 90° angle between excitation anddetection paths assures that a minimum amount of excitation radiationentering through the wall 19A will exit through wall 19B. In addition,the 90° angle permits a maximum amount of emitted light, e.gfluorescence, to be collected through wall 19B. The walls 19A, 19B arepreferably joined to each other to form a “V” shaped intersection at thebottom of the chamber 17. Alternatively, the angled walls 19A, 19B neednot be directly joined to each other, but may be separated by anintermediary portion, such as another wall or various mechanical orfluidic features which do not interfere with the thermal and opticalperformance of the vessel. For example, the walls 19A, 19B may meet at aport which leads to another processing area in communication with thechamber 17, such as an integrated capillary electrophoresis area. In thepresently preferred embodiment, a locating tab 27 extends from the frame16 below the intersection of walls 19A, 19B. The locating tab 27 is usedto properly position the vessel 12 in a heat-exchanging module describedbelow with reference to FIG. 6.

Optimum optical sensitivity may be attained by maximizing the opticalpath length of the light beams exciting the labeled analytes in thesample and the emitted light that is detected, as represented by theequation:I _(o) /I _(i) =C*L*A,

where I_(o) is the illumination output of the emitted light in volts,photons or the like, C is the concentration of analyte to be detected,I_(i) is the input illumination, L is the path length, and A is theintrinsic absorptivity of the dye used to label the analyte.

The thin, flat reaction vessel 12 of the present invention optimizesdetection sensitivity by providing maximum optical path length per unitanalyte volume. Referring to FIGS. 3–4, the vessel 12 is preferablyconstructed such that each of the sides walls 19A, 19B, 20A, 20B of thechamber 17 has a length L in the range of 1 to 15 mm, the chamber has awidth W in the range of 1.4 to 20 mm, the chamber has a thickness T inthe range of 0.5 to 5 mm, and the ratio of the width W of the chamber tothe thickness T of the chamber is at least 2:1. These parameters arepresently preferred to provide a vessel having a relatively largeaverage optical path length through the chamber, i.e. 1 to 15 mm onaverage, while still keeping the chamber sufficiently thin to allow forextremely rapid heating and cooling of the sample contained therein. Theaverage optical path length of the chamber 17 is the distance from thecenter of the side wall 19A to the center of the chamber 17 plus thedistance from the center of the chamber 17 to the center of the sidewall 19B. The thickness T of the chamber 17 is defined by the thicknessof the sides walls 19A, 19B, 20A, 20B.

More preferably, the vessel 12 is constructed such that each of thesides walls 19A, 19B, 20A, 20B of the chamber 17 has a length L in therange of 5 to 12 mm, the chamber has a width W in the range of 7 to 17mm, the chamber has a thickness T less than or equal to 3 mm, and theratio of the width W of the chamber to the thickness T of the chamber isat least 4:1. These ranges are more preferable because they provide avessel having both a larger average optical path length (i.e., 5 to 12mm) and volume capacity (i.e., 10 to 430 microliters) while stillmaintaining a chamber sufficiently thin to permit extremely rapidheating and cooling of a sample. The relatively large volume capacityprovides for increased sensitivity in the detection of low concentrationanalytes, such as nucleic acids.

In the presently preferred embodiment, the reaction vessel 12 has adiamond-shaped chamber 17 defined by the side walls 19A, 19B, 20A, 20B,each of the side walls has a length of about 10 mm, the chamber has awidth of about 14 mm, the chamber has a thickness T of 1 mm as definedby the thickness of the frame 16, and the chamber has a volume capacityof about 100 microliters. This reaction vessel provides a relativelylarge average optical path length of 10 mm through the chamber 17.Additionally, the thin chamber allows for extremely rapid heating and/orcooling of the sample contained therein. The diamond-shape of thechamber 17 helps prevent air bubbles from forming in the chamber as itis filled with the sample and also aids in optical interrogation of thesample.

The frame 16 is preferably made of an optically transmissive material,e.g., a polycarbonate or clarified polypropylene, so that the side walls19A, 19B are optically transmissive. As used herein, the term opticallytransmissive means that one or more wavelengths of light may betransmitted through the walls. In the preferred embodiment, theoptically transmissive walls 19A, 19B are substantially transparent. Inaddition, one or more optical elements may be present on the opticallytransmissive side walls 19A, 19B. The optical elements may be designed,for example, to maximize the total volume of solution which isilluminated by a light source, to focus excitation light on a specificregion of the chamber 17, or to collect as much fluorescence signal fromas large a fraction of the chamber volume as possible. In alternativeembodiments, the optical elements may comprise gratings for selectingspecific wavelengths, filters for allowing only certain wavelengths topass, or colored lenses to provide filtering functions. The wallsurfaces may be coated or comprise materials such as liquid crystal foraugmenting the absorption of certain wavelengths. In the presentlypreferred embodiment, the optically transmissive walls 19A, 19B aresubstantially clear, flat windows having a thickness of about 1 mm.

As shown in FIG. 2, the side walls 20A, 20B preferably includesreflective faces 21 which internally reflect light trying to exit thechamber 17 through the side walls 20A, 20B. The reflective faces 21 arearranged such that adjacent faces are angularly offset from each otherby about 90°. In addition, the frame 16 defines open spaces between theside walls 20A, 20B and support ribs 15. The open spaces are occupied byambient air that has a different refractive index than the materialcomposing the frame (e.g., plastic). Due to the difference in therefractive indexes, the reflective faces 21 are effective for internallyreflecting light trying to exit the chamber 17 through the walls 20A,20B and provide for increased detection of optical signal through thewalls 19A, 19B. In the preferred embodiment, the optically transmissiveside walls 19A, 19B define the bottom portion of the diamond-shapedchamber 17, and the retro-reflective side walls 20A, 20B define the topportion of the chamber.

The reaction vessel 12 may be used in manual operations performed byhuman technicians or in automated operations performed by machines, e.g.pick-and-place machines. As shown in FIG. 2, for automated embodiments,the cap 36 preferably includes a tapered engagement aperture 46 forreceiving and establishing a fit with a robotic arm or machine tip (notshown in FIG. 2), thus enabling the machine tip to pick and place theplug 22 into the aperture 14. The engagement aperture 46 preferably hastapered side walls for establishing a friction fit with the machine tip.Alternatively, the engagement aperture 46 may be designed to establish avacuum fit with the machine tip. The cap 36 may optionally includealignment apertures 48A, 48B used by the machine tip to properly alignthe cap 36 as the plug 22 is inserted into the aperture 14.

A preferred method for fabricating the reaction vessel 12 will now bedescribed with reference to FIGS. 1–2A. The reaction vessel 12 may befabricated by first molding the rigid frame 16 and the loading structure30 using known injection molding techniques. The frame 16 and loadingstructure 30 are preferably molded as a single piece of polymericmaterial, e.g., clarified polypropylene. The channel 39 is preferablyproduced with at least one narrow cross-sectional dimension to controlfluid flow as previously described. In embodiments in which the vesselincludes a valve 11 in the channel 39 (FIG. 2B) or screen or membrane 13in the channel 39 (FIG. 2C), the valve or membrane can also beintegrally molded in one piece with the frame 16 and loading structure30. This one-piece molding technique is suitable for forming a flapvalve or piercable or burstable membrane in the channel 39.Alternatively, the vessel can be insert molded by placing a valve,screen, or membrane in the mold and molding the frame and loadingstructure around the inserted piece to produce a vessel with a valve,screen, or membrane in the channel 39. Alternatively, a valve, screen,or membrane may simply be inserted into the channel 39 after molding theframe 16 and loading structure 30.

Referring again to FIGS. 1–2A, after the frame and loading structure areproduced, thin, flexible sheets are cut to size and sealed to oppositesides of the frame 16 to form the major walls 18 of the chamber 17. Themajor walls 18 are preferably cast or extruded films of polymericmaterial, e.g., polypropylene films, that are cut to size and attachedto the frame 16 using the following procedure. A first piece of film isplaced over one side of the bottom portion of the frame 16. The frame 16preferably includes a tack bar 57 (FIG. 2) for aligning the top edge ofthe film. The film is placed over the bottom portion of the frame 16such that the top edge of the film is aligned with the tack bar 57 andsuch that the film completely covers the bottom portion of the frame 16below the tack bar 57. The film should be larger than the bottom portionof the frame 16 so that it may be easily held and stretched flat acrossthe frame. The film is then cut to size to match the outline of theframe by clamping to the frame the portion of the film that covers theframe and cutting away the portions of the film that extend past theperimeter of the frame using, e.g., a laser or die. The film is thentack welded to the frame, preferably using a laser.

The film is then sealed to the frame 16, preferably by heat sealing.Heat sealing is presently preferred because it produces a strong sealwithout introducing potential contaminants to the vessel as the use ofadhesive or solvent bonding techniques might do. Heat sealing is alsosimple and inexpensive. At a minimum, the film should be completelysealed to the surfaces of the side walls 19A, 19B, 20A, 20B. Morepreferably, the film is additionally sealed to the surfaces of thesupport ribs 15 and tack bar 57. The heat sealing may be performedusing, e.g., a heated platen. An identical procedure may be used to cutand seal a second sheet to the opposite side of the frame 16 to form theother major wall of the chamber 17.

Many variations to this fabrication procedure are possible. For example,in an alternative embodiment, the film is stretched across the bottomportion of the frame 16 and then sealed to the frame prior to cuttingthe film to size. After sealing the film to the frame, the portions ofthe film that extend past the perimeter of the frame arc cut away using,e.g., a laser or die. Although it is presently preferred to mold theframe 16 as a single piece, it is also possible to fabricate the framefrom multiple pieces. For example, the side walls 19A, 19B forming theangled optical windows may be molded from polycarbonate, which has goodoptical transparency, while the rest of the frame is molded frompolypropylene, which is inexpensive and compatible with PCR. Theseparate pieces can be attached together in a secondary step. Forexample, the side walls 19A, 19B may be press-fitted and/or bonded tothe remaining portion of the frame 16. The flexible walls 18 may then beattached to opposite sides of the frame 16 as previously described.

The plug 22 is preferably produced using known injection moldingtechniques. In the preferred embodiment, the plug 22 is molded as aone-piece part of elastomeric material. Suitable elastomers from whichthe plug may be molded include thermalplastic elastomers (e.g.,Santoprene® commercially available from the Monsanto Company of St.Louis, Mo.). Alternatively, the plug 22 may be produced by molding aplastic body and placing an elastomeric ring (e.g., an o-ring) aroundthe plastic body to form the sealing ring 32.

Referring again to FIG. 4, the plates 50A, 50B may be made of variousthermally conductive materials including ceramics or metals. Suitableceramic materials include aluminum nitride, aluminum oxide, berylliumoxide, and silicon nitride. Other materials from which the plates may bemade include, e.g., gallium arsenide, silicon, silicon nitride, silicondioxide, quartz, glass, diamond, polyacrylics, polyamides,polycarbonates, polyesters, polyimides, vinyl polymers, and halogenatedvinyl polymers, such as polytetrafluoroethylenes. Other possible platematerials include chrome/aluminum, superalloys, zircaloy, aluminum,steel, gold, silver, copper, tungsten, molybdenum, tantalum, brass,sapphire, or any of the other numerous ceramic, metal, or polymericmaterials available in the art.

Ceramic plates are presently preferred because their inside surfaces maybe conveniently machined to very high smoothness for high wearresistance, high chemical resistance, and good thermal contact to theflexible walls of the reaction vessel. Ceramic plates can also be madevery thin, preferably between about 0.5 and 1 mm, for low thermal massto provide for extremely rapid temperature changes. A plate made fromceramic is also both a good thermal conductor and an electricalinsulator, so that the temperature of the plate may be well controlledusing a resistive heating element coupled to the plate.

Various thermal elements may be employed to heat and/or cool the plates50A, 50B and thus control the temperature of the sample in the chamber17. In general, suitable heating elements for heating the plate includeconductive heaters, convection heaters, or radiation heaters. Examplesof conductive heaters include resistive or inductive heating elementscoupled to the plates, e.g., resistors or thermoelectric devices.Suitable convection heaters include forced air heaters or fluidheat-exchangers for flowing fluids past the plates. Suitable radiationheaters include infrared or microwave heaters. Similarly, variouscooling elements may be used to cool the plates. For example, variousconvection cooling elements may be employed such as a fan, Peltierdevice, refrigeration device, or jet nozzle for flowing cooling fluidspast the surfaces of the plates. Alternatively, various conductivecooling elements may be used, such as a heat sink, e.g. a cooled metalblock, in direct contact with the plates.

Referring to FIG. 5, in the preferred embodiment a resistive heatingelement 56 is coupled to each plate 50. The resistive heating element 56is preferably a thick or thin film and may be directly screen printedonto each plate 50, particularly plates comprising a ceramic material,such as aluminum nitride or aluminum oxide. Screen-printing provideshigh reliability and low cross-section for efficient transfer of heatinto the reaction chamber. Thick or thin film resistors of varyinggeometric patterns may be deposited on the outer surfaces of the platesto provide more uniform heating, for example by having denser resistorsat the extremities and thinner resistors in the middle. Although it ispresently preferred to deposit a heating element on the outer surface ofeach plate, a heating element may alternatively be baked inside of eachplate, particularly if the plates are ceramic. The heating element 56may comprise metals, tungsten, polysilicon, or other materials that heatwhen a voltage difference is applied across the material.

The heating element 56 has two ends which are connected to respectivecontacts 54 which are in turn connected to a voltage source (not shownin FIG. 5) to cause a current to flow through the heating element 56.Each plate 50 also preferably includes a temperature sensor 52, such asa thermocouple, thermistor, or RTD, which is connected by two traces 53to respective contacts 54. The temperature sensor 52 may be used tomonitor the temperature of the plate 50 in a controlled feedback loop.

The plates preferably have a low thermal mass to enable rapid heatingand cooling of the plates. In particular, it is presently preferred thateach of the plates has a thermal mass less than about 5 J/° C., morepreferably less than 3 J/° C., and most preferably less than 1 J/° C. Asused herein, the term thermal mass of a plate is defined as the specificheat of the plate multiplied by the mass of the plate. In addition, eachplate should be large enough to cover a respective major wall of thereaction chamber. In the presently preferred embodiment, for example,each of the plates has a width X in the range of 2 to 22 mm, a length Yin the range of 2 to 22 mm, and a thickness in the range of 0.5 to 5 mm.The width X and length Y of each plate is selected to be slightly largerthan the width and length of the reaction chamber. Moreover, each platepreferably has an angled bottom portion matching the geometry of thebottom portion of the reaction chamber, as is described below withreference to FIG. 12. Also in the preferred embodiment, each of theplates is made of aluminum nitride having a specific heat of about 0.75J/g ° C. The mass of each plate is preferably in the range of 0.005 to1.3 g so that each plate has a thermal mass in the range of 0.00375 to 1J/° C.

FIG. 6 is a schematic side view of a heat-exchanging module 60 intowhich the reaction vessel 12 is inserted for thermal processing andoptical interrogation. The module 60 preferably includes a housing 62for holding the various components of the module. The module 60 alsoincludes the thermally conductive plates 50 described above. The housing62 includes a slot (not shown in FIG. 6) above the plates 50 so that thereaction chamber of the vessel 12 may be inserted through the slot andbetween the plates. The heat-exchanging module 60 also preferablyincludes a cooling system, such as a fan 66. The fan 66 is positioned toblow cooling air past the surfaces of the plates 50 to cool the platesand hence cool the sample in the vessel 12. The housing 62 preferablydefines channels for directing the cooling air past the plates 50 andout of the module 60.

The heat-exchanging module 60 further includes an optical excitationassembly 68 and an optical detection assembly 70 for opticallyinterrogating the sample contained in the vessel 12. The excitationassembly 68 includes a first circuit board 72 for holding its electroniccomponents, and the detection assembly 68 includes a second circuitboard 74 for holding its electronic components. The excitation assembly68 includes one or more light sources, such as LEDs, for excitingfluorescently-labeled analytes in the vessel 12. The excitation assembly68 also includes one or more lenses for collimating the light from thelight sources, as well as filters for selecting the excitationwavelength ranges of interest. The detection assembly 70 includes one ormore detectors, such as photodiodes, for detecting the light emittedfrom the vessel 12. The detection assembly 70 also includes one or morelenses for focusing and collimating the emitted light, as well asfilters for selecting the emission wavelength ranges of interest. Thespecific components of the optics assemblies 68, 70 are described ingreater detail below with reference to FIGS. 14–17.

The optics assemblies 68, 70 are positioned in the housing 62 such thatwhen the chamber of the vessel 12 is inserted between the plates 50, thefirst optics assembly 68 is in optical communication with the chamber 17through the optically transmissive side wall 19A (see FIG. 2) and thesecond optics assembly 70 is in optical communication with the chamberthrough the optically transmissive side wall 19B (FIG. 2). In thepreferred embodiment, the optics assemblies 68, 70 are placed intooptical communication with the optically transmissive side walls bysimply locating the optics assemblies 68, 70 next to the bottom edges ofthe plates 50 so that when the chamber of the vessel is placed betweenthe plates, the optics assemblies 68, 70 directly contact, or are inclose proximity to, the side walls.

As shown in the partially cut-away view of FIG. 12 (in which the topportion of the vessel 12 has been cut away), the vessel 12 has an angledbottom portion (e.g., triangular) formed by the optically transmissiveside walls 19A, 19B. Each of the plates 50A, 50B has a correspondinglyshaped bottom portion. The bottom portion of the first plate 50A has afirst bottom edge 98A and a second bottom edge 98B. Similarly, thebottom portion of the second plate 50B has a first bottom edge 99A and asecond bottom edge 99B. The first and second bottom edges of each plateare preferably angularly offset from each other by the same angle thatthe side walls 19A, 19B are offset from each other (e.g., 90°).Additionally, the plates 50A, 50B are preferably positioned to receivethe chamber of the vessel 12 between them such that the first side wall19A is positioned substantially adjacent and parallel to each of thefirst bottom edges 98A, 99A and such that the second side wall 19B ispositioned substantially adjacent and parallel to each of the secondbottom edges 98B, 99B. This arrangement provides for easy optical accessto the optically transmissive side walls 19A, 19B and hence to thechamber of the vessel 12.

The side walls 19A, 19B may be positioned flush with the edges of theplates 50A, 50B, or more preferably, the side walls 19A, 19B may bepositioned such that they protrude slightly past the edges of theplates. As is explained below with reference to FIGS. 14–17, each opticsassembly preferably includes a lens that physically contacts arespective one of the side walls 19A, 19B. It is preferred that the sidewalls 19A, 19B protrude slightly (e.g., 0.02 to 0.3 mm) past the edgesof the plates 50A, 50B so that the plates do not physically contact anddamage the lenses. A gel or fluid may optionally be used to establish orimprove optical communication between each optics assembly and the sidewalls 19A, 19B. The gel or fluid should have a refractive index close tothe refractive indexes of the elements that it is coupling.

Referring again to FIG. 6, the optics assemblies 68, 70 are preferablyarranged to provide a 90° angle between excitation and detection paths.The 90° angle between excitation and detection paths assures that aminimum amount of excitation radiation entering through the first sidewall of the chamber exits through the second side wall. Also, the 90°angle permits a maximum amount of emitted radiation to be collectedthrough the second side wall. In the preferred embodiment, the vessel 12includes a locating tab 27 (see FIG. 2) that fits into a slot formedbetween the optics assemblies 68, 70 to ensure proper positioning of thevessel 12 for optical detection. For improved detection, the module 60also preferably includes a light-tight lid (not shown) that is placedover the top of the vessel 12 and made light-tight to the housing 62after the vessel is inserted between the plates 50.

Although it is presently preferred to locate the optics assemblies 68,70 next to the bottom edges of the plates 50, many other arrangementsare possible. For example, optical communication may be establishedbetween the optics assemblies 68, 70 and the walls of the vessel 12 viaoptical fibers, light pipes, wave guides, or similar devices. Oneadvantage of these devices is that they eliminate the need to locate theoptics assemblies 68, 70 physically adjacent to the plates 50. Thisleaves more room around the plates in which to circulate cooling air orrefrigerant, so that cooling may be improved.

The heat-exchanging module 60 also includes a PC board 76 for holdingthe electronic components of the module and an edge connector 80 forconnecting the module 60 to a base instrument, as will be describedbelow with reference to FIG. 18. The heating elements and temperaturesensors on the plates 50, as well as the optical boards 72, 74, areconnected to the PC board 76 by flex cables (not shown in FIG. 6 forclarity of illustration). The module. 60 may also include a groundingtrace 78 for shielding the optical detection circuit. The module 60 alsopreferably includes an indicator, such as an LED 64, for indicating to auser the current status of the module such as “ready to load samplc”,“ready to load reagent.” “heating,” “cooling,” “finished,” or “fault”.

The housing 62 may be molded from a rigid, high-performance plastic, orother conventional material. The primary functions of the housing 62 areto provide a frame for holding the plates 50, optics assemblies 68, 70,fan 66, and PC board 76. The housing 62 also preferably provides flowchannels and ports for directing cooling air from the fan 66 across thesurfaces of the plates 50 and out of the housing. In the preferredembodiment, the housing 62 comprises complementary pieces (only onepiece shown in the schematic side view of FIG. 6) that fit together toenclose the components of the module 60 between them.

The opposing plates 50 are positioned to receive the chamber of thevessel 12 between them such that the flexible major walls of the chambercontact and conform to the inner surfaces of the plates. It is presentlypreferred that the plates 50 be held in an opposing relationship to eachother using, e.g., brackets, supports, or retainers. Alternatively, theplates 50 may be spring-biased towards each other as described inInternational Publication Number WO 98/38487, the disclosure of which isincorporated by reference herein. In another embodiment of theinvention, one of the plates is held in a fixed position, and the secondplate is spring-biased towards the first plate. If one or more springsare used to bias the plates towards each other, the springs should besufficiently stiff to ensure that the plates are pressed against theflexible walls of the vessel with sufficient force to cause the walls toconform to the inner surfaces of the plates.

FIGS. 7–8 illustrate a preferred support structure 81 for holding theplates 50A, 50B in an opposing relationship to each other. FIG. 7 showsan exploded view of the structure, and FIG. 8 shows an assembled view ofthe structure. For clarity of illustration, the support structure 81 andplates 50A, 50B are shown upside down relative to their normalorientation in the heat-exchanging module of FIG. 6. Referring to FIG.7, the support structure 81 includes a mounting plate 82 having a slot83 formed therein. The slot 83 is sufficiently large to enable thechamber of the vessel to be inserted through it. Spacing posts 84A, 84Bextend from the mounting plate 82 on opposite sides of the slot 83.Spacing post 84A has indentations 86 formed on opposite sides thereof(only one side visible in the isometric view of FIG. 7), and spacingpost 84B has indentations 87 formed on opposite sides thereof (only oneside visible in the isometric view of FIG. 7). The indentations 86, 87in the spacing posts are for receiving the edges of the plates 50A, 50B.To assemble the structure, the plates 50A, 50B are placed againstopposite sides of the spacing posts 84A, 84B such that the edges of theplates are positioned in the indentations 86, 87. The edges of theplates are then held in the indentations using a suitable retentionmeans. In the preferred embodiment, the plates are retained by retentionclips 88A, 88B. Alternatively, the plates 50A, 50B may be retained byadhesive bonds, screws, bolts, clamps, or any other suitable means.

The mounting plate 82 and spacing posts 84A, 84B are preferablyintegrally formed as a single molded piece of plastic. The plasticshould be a high temperature plastic, such as polyetherimide, which willnot deform of melt when the plates 50A, 50B are heated. The retentionclips 84A, 84B are preferably stainless steel. The mounting plate 82 mayoptionally include indentations 92A, 92B for receiving flex cables 90A,90B, respectively, that connect the heating elements and temperaturesensors disposed on the plates 50A, 50B to the PC board 76 of theheat-exchanging module 60 (FIG. 6). The portion of the flex cables 90Aadjacent the plate 50A is held in the indentation 92A by a piece of tape94A, and the portion of the flex cables 90B adjacent the plate 50B isheld in the indentation 92B by a piece of tape 94B.

FIG. 9 is an isometric view of the assembled support structure 81. Themounting plate 82 preferably includes tabs 96 extending from oppositesides thereof for securing the structure 81 to the housing of theheat-exchanging module. Referring again to FIG. 6, the housing 62preferably includes slots for receiving the tabs to hold the mountingplate 82 securely in place. Alternatively, the mounting plate 82 may beattached to the housing 62 using, e.g., adhesive bonding, screws, bolts,clamps, or any other conventional means of attachment.

Referring again to FIG. 7, the support structure 81 preferably holds theplates 50A, 50B so that their inner surfaces are angled very slightlytowards each other. In the preferred embodiment, each of the spacingposts 84A, 84B has a wall 89 that is slightly tapered so that when theplates 50A, 50B are pressed against opposite sides of the wall, theinner surfaces of the plates are angled slightly towards each other. Asbest shown in FIG. 4, the inner surfaces of the plates 50A, 50B angletowards each other to form a slightly V-shaped slot into which thechamber 17 is inserted. The amount by which the inner surfaces areangled towards each other is very slight, preferably about 1° fromparallel. The surfaces are angled towards each other so that, prior tothe insertion of the chamber 17 between the plates 50A, 50B, the bottomsof the plates are slightly closer to each other than the tops. Thisslight angling of the inner surfaces enables the chamber 17 of thevessel to be inserted between the plates and withdrawn from the platesmore easily. Alternatively, the inner surfaces of the plates 50A, 50Bcould be held parallel to each other, but insertion and removal of thevessel 12 may be more difficult.

In addition, the inner surfaces of the plates 50A, 50B are preferablyspaced from each other a distance equal to the thickness of the frame16. In embodiments in which the inner surfaces are angled towards eachother, the centers of the inner surfaces are preferably spaced adistance equal to the thickness of the frame 16 and the bottoms of theplates are initially spaced a distance that is slightly less than thethickness of the frame 16. When the chamber 17 is inserted between theplates 50A, 50B, the rigid frame 16 forces the bottom portions of theplates apart so that the chamber 17 is firmly sandwiched between theplates. The distance that the plates 50A, 50B are wedged apart by theframe 16 is usually very small, e.g., about 0.035 mm if the thickness ofthe frame is 1 mm and the inner surfaces are angled towards each otherby 1°.

Referring again to FIG. 8, the retention clips 88A, 88B should besufficiently flexible to accommodate this slight outward movement of theplates 50A, 50B, yet sufficiently stiff to hold the plates within therecesses in the spacing posts 84A, 84B during insertion and removal ofthe vessel. The wedging of the vessel between the plates 50A, 50Bprovides an initial preload against the chamber and ensures that theflexible major walls of the chamber, when pressurized, establish goodthermal contact with the inner surfaces of the plates.

Referring again to FIG. 6, to limit the amount that the plates 50 canspread apart due to the pressurization of the vessel 12, stops may bemolded into the housings of optics assemblies 68, 70. As shown in FIG.10, the housing 221 of the optics assembly 70 includes claw-like stops247A, 247B that extend outwardly from the housing. As shown in FIG. 11,the housing 221 is positioned such that the bottom edges of the plates50A, 50B are inserted between the stops 247A, 247B. The stops 247A, 247Bthus prevent the plates 50A, 50B from spreading farther than apredetermined maximum distance from each other. Although not shown inFIG. 11 for illustrative clarity, the optics assembly 68 (see FIG. 6)has a housing with corresponding stops for preventing the other halvesof the plates 50A, 50B from spreading farther than the predeterminedmaximum distance from each other. Referring again to FIG. 11, themaximum distance that stops 247A, 247B permit the inner surfaces of theplates 50A, 50B to be spaced from each other should closely match thethickness of the frame 16. Preferably, the maximum spacing of the innersurfaces of the plates 50A, 50B is slightly larger than the thickness ofthe frame 16 to accommodate tolerance variations in the vessel 12 andplates 50A, 50B. For example. the maximum spacing is preferably about0.1 to 0.3 mm greater than the thickness of the frame 16.

FIGS. 13A and 13B show the fluorescent excitation and emission spectra,respectively, of four fluorescent dyes of interest. These dyes arestandard fluorescent dyes used with the TaqMan® chemistry (availablefrom the Perkin-Elmer Corporation, Foster City, Calif.) and are wellknown by their acronyms FAM, TET, TAMRA, and ROX. Although the preferredembodiment is described with reference to these four dyes, it is to beunderstood that the system of the present invention is not limited tothese particular dyes or to the TaqMan® chemistry. The system may beused with any fluorescent dyes or with interculating dyes such asSYBRGreen™ or ethidium bromide. Such dyes are commercially availablefrom various well known suppliers. Fluorescent dyes, probes, andlabeling chemistries for labeling analytes in a sample are well known inthe art and need not be discussed further herein. Further, althoughfluorescence detection is presently preferred, the system of the presentinvention is not limited to detection based upon fluorescent labels. Thesystem may be applicable to detection based upon phosphorescent labels,chemiluminescent labels, or electrochemiluminescent labels.

As shown in FIG. 13A, the excitation spectra curves for FAM, TET, TAMRA,and ROX are typically very broad at the base, but sharper at the peaks.As shown in FIG. 13B, the relative emission spectra curves for the samedyes are also very broad at the base and sharper at the peaks. Thus,these dyes have strongly overlapping characteristics in both theirexcitation and emission spectra. The overlapping characteristics havetraditionally made it difficult to distinguish the fluorescent signal ofone dye from another when multiple dyes are used to label differentanalytes in a sample.

Preferably, multiple light sources are used to provide excitation beamsto the dyes in multiple excitation wavelength ranges. Each light sourceprovides excitation light in a wavelength range matched to the peakexcitation range of a respective one of the dyes. In the preferredembodiment, the light sources are blue and green LEDs. FIG. 13C showsthe effects of filtering the outputs of blue and green LEDs to providesubstantially distinct excitation wavelength ranges. Typical blue andgreen LEDs have substantial overlap in the range of around 480 nmthrough 530 nm. By the filtering regime of the present invention, theblue LED light is filtered to a range of about 450 to 495 nm to matchthe relative excitation peak for FAM. The green LED light is filtered toa first range of 495 to 527 nm corresponding to the excitation peak forTET, a second range of 527 to 555 nm corresponding to the excitationpeak for TAMRA, and a third range of 555 to 593 nm corresponding to theexcitation peak for ROX.

FIG. 13D shows the effects of filtering light emitted (fluorescentemission) from each of the four dyes to form distinct emissionwavelength ranges. As shown previously in FIG. 13B, the fluorescentemissions of the dyes before filtering are spherically diffuse withoverlapping spectral bandwidths, making it difficult to distinguish thefluorescent output of one dye from another. As shown in FIG. 13D, byfiltering the fluorescent emissions of the dyes into substantiallydistinct wavelength ranges, a series of relatively narrow peaks(detection windows) are obtained, making it possible to distinguish thefluorescent outputs of different dyes, thus enabling the detection of anumber of different fluorescently-labeled analytes in a sample.

FIG. 14 is a schematic, plan view of the optical excitation assembly 68.The assembly 68 is positioned adjacent the reaction vessel 12 totransmit excitation beams to the sample contained in the chamber 17.FIG. 15 is an exploded view of the excitation assembly. As shown inFIGS. 14–15, the excitation assembly 68 includes a housing 219 forholding various components of the assembly. The housing 219 preferablycomprises one or more molded pieces of plastic. In the preferredembodiment, the housing 219 is a multi-part housing comprised of threehousing elements 220A, 220B, and 220C. The upper and lower housingelements 220A and 220C are preferably complementary pieces that coupletogether and snap-fit into housing element 220B. In this embodiment, thehousing elements 220A and 220C are held together by screws 214. Inalternative embodiments, the entire housing 219 may be a one-piecehousing that holds a slide-in optics package.

The lower housing element 220C includes an optical window 235 into whichis placed a cylindrical rod lens 215 for focusing excitation beams intothe chamber 17. In general, the optical window 235 may simply comprisean opening in the housing through which excitation beams may betransmitted to the chamber 17. The optical window may optionally includean optically transmissive or transparent piece of glass or plasticserving as a window pane, or as in the preferred embodiment, a lens forfocusing excitation beams. The lens 215 preferably directly contacts oneof the optically transmissive side walls of the chamber 17.

The optics assembly 68 also includes four light sources, preferably LEDs100A, 100B, 100C, and 100D, for transmitting excitation beams throughthe lens 215 to the sample contained in the chamber 17. In general, eachlight source may comprise a laser, a light bulb, or an LED. In thepreferred embodiment, each light source comprises a pair of directionalLEDs. In particular, the four light sources shown in FIGS. 14–15 arepreferably a first pair of green LEDs 100A, a second pair of green LEDs100B, a pair of blue LEDs 100C, and a third pair of green LEDs 100D. TheLEDs receive power through leads 201 which are connected to a powersource (not shown in FIGS. 14–15). The LEDs are mounted to the opticalcircuit board 72 which is attached to the back of the housing element220B so that the LEDs are rigidly fixed in the housing. The opticalcircuit board 72 is connected to the main PC board of theheat-exchanging module (shown in FIG. 6) via the flex cable 103.

The optics assembly 68 further includes a set of filters and lensesarranged in the housing 219 for filtering the excitation beams generatedby the LEDs so that each of the beams transmitted to the chamber 17 hasa distinct excitation wavelength range. As shown in FIG. 15, the lowerhousing element 220C preferably includes walls 202 that create separateexcitation channels in the housing to reduce potential cross-talkbetween the different pairs of LEDs. The walls 202 preferably includeslots for receiving and rigidly holding the filters and lenses. Thefilters and lenses may also be fixed in the housing by means of anadhesive used alone, or more preferably, with an adhesive used incombination with slots in the housing.

Referring to FIG. 14, the filters in the optics assembly 68 may beselected to provide excitation beams to the sample in the chamber 17 inany desired excitation wavelength ranges. The optics assembly 68 maytherefore be used with any fluorescent, phosphorescent,chemiluminescent, or electrochemiluminescent labels of interest. Forpurposes of illustration, one specific embodiment of the assembly 68will now be described in which the assembly is designed to provideexcitation beams corresponding to the peak excitation wavelength rangesFAM, TAMRA, TET, and ROX.

In this embodiment, a pair of 593 nm low pass filters 203 are positionedin front of green LEDs 100A, a pair of 555 nm low pass filters 204 arepositioned in front of green LEDs 100B, a pair of 495 nm low passfilters 205 are positioned in front of blue LEDs 100C, and a pair of 527nm low pass filters 206 are positioned in front of green LEDs 100D.Although it is presently preferred to position a pair of low passfilters in front of each pair of LEDs for double filtering of excitationbeams, a single filter may be used in alternative embodiments. Inaddition, a lens 207 is preferably positioned in front of each pair offilters for collimating the filtered excitation beams. The opticsassembly 68 also includes a 495 nm high pass reflector 208, a 527 nmhigh pass reflector 209, a mirror 210, a 555 nm low pass reflector 211,and a 593 nm low pass reflector 212. The reflecting filters and mirrors208–212 are angularly offset by 30° from the low pass filters 203–206.

The excitation assembly 68 transmits excitation beams to the chamber 17in four distinct excitation wavelength ranges as follows. When the greenLEDs 100A are activated, they generate an excitation beam that passesthrough the pair of 593 nm low pass filters 203 and through the lens207. The excitation beam then reflects off of the 593 nm low passreflector 212, passes through the 555 nm low pass reflector 211,reflects off of the 527 nm high pass reflector 209, and passes throughthe lens 215 into the reaction chamber 17. The excitation beam from theLEDs 100A is thus filtered to a wavelength range of 555 to 593 nmcorresponding to the peak excitation range for ROX. When the green LEDs100B are activated, they generate an excitation beam that passes throughthe pair of 555 nm low pass filters 204, reflects off of the 555 nm lowpass reflector 211, reflects off of the 527 nm high pass reflector 209,and passes through the lens 215 into the reaction chamber 17. Theexcitation beam from LEDs 100B is thus filtered to a wavelength range of527 to 555 nm corresponding to the peak excitation range for TAMRA.

When the blue LEDs 100C are activated, they generate an excitation beamthat passes through the pair of 495 nm low pass filters 205, through the495 nm high pass reflector 208, through the 527 nm high pass reflector209, and through the lens 215 into the reaction chamber 17. Theexcitation beam from LEDs 100C is thus filtered to a wavelength below495 nm corresponding to the peak excitation range for FAM. When thegreen LEDs 100D are activated, they generate an excitation beam thatpasses through the pair of 527 nm low pass filters 206, reflects off ofthe mirror 210, reflects off of the 495 nm high pass reflector 208,passes through the 527 nm high pass reflector 209, and passes throughthe lens 215 into the reaction chamber 17. The excitation beam from LEDs100D is thus filtered to a wavelength range of 495 to 527 nmcorresponding to the peak excitation range for TET. In operation, theLEDs 100A, 100B, 100C, 100D are sequentially activated to excite thedifferent fluorescent labels contained in the chamber 17 with excitationbeams in substantially distinct wavelength ranges.

FIG. 16 is a schematic, plan view of the optical detection assembly 70.The assembly 70 is positioned adjacent the reaction vessel 12 to receivelight emitted from the chamber 17. FIG. 17 is an exploded view of thedetection assembly 70. As shown in FIGS. 16–17, the assembly 70 includesa housing 221 for holding various components of the assembly. Thehousing 221 preferably comprises one or more molded plastic pieces. Inthe preferred embodiment, the housing 221 is a multi-part housingcomprised of upper and lower housing elements 234A and 234B. The housingelements 234A, 234B are complementary, mating pieces that are heldtogether by screws 214. In alternative embodiments, the entire housing221 may be a one-piece housing that holds a slide-in optics package.

The lower housing element 234B includes an optical window 237 into whichis placed a cylindrical rod lens 232 for collimating light emitted fromthe chamber 17. In general, the optical window may simply comprise anopening in the housing through which the emitted light may be received.The optical window may optionally include an optically transmissive ortransparent piece of glass or plastic serving as a window pane, or as inthe preferred embodiment, the lens 232 for collimating light emittedfrom the chamber 17. The lens 232 preferably directly contacts one ofthe optically transmissive side walls of the chamber 17.

The optics assembly 70 also includes four detectors 102A, 102B. 102C,and 102D for detecting light emitted from the chamber 17 that isreceived through the lens 232. In general, each detector may be aphotomultiplier tube, CCD, photodiode, or other known detector. In thepreferred embodiment, each detector is a PIN photodiode. The detectors102A, 102B. 102C, and 102D are preferably rigidly fixed in recessesformed in the lower housing element 234B. The detectors are electricallyconnected by leads 245 to the optical circuit board 74 (see FIG. 6)which is preferably mounted to the underside of the lower housingelement 234B.

The optics assembly 70 further includes a set of filters and lensesarranged in the housing 221 for separating light emitted from thechamber 17 into different emission wavelength ranges and for directingthe light in each of the emission wavelength ranges to a respective oneof the detectors. As shown in FIG. 17, the lower housing element 234Bpreferably includes walls 247 that create separate detection channels inthe housing, with one of the detectors positioned at the end of eachchannel. The walls 247 preferably include slots for receiving andrigidly holding the filters and lenses. The filters and lenses may alsobe rigidly fixed in the housing 221 by an adhesive used alone, or morepreferably, with an adhesive used in combination with slots in thehousing.

Referring to FIG. 16, the filters in the optics assembly 70 may beselected to block light emitted from the chamber 17 outside of anydesired emission wavelength ranges. The optics assembly 70 may thereforebe used with any fluorescent, phosphorescent, chemiluminescent, orelectrochemiluminescent labels of interest. For purposes ofillustration, one specific embodiment of the assembly 70 will now bedescribed in which the assembly is designed to detect light emitted fromthe chamber 17 in the peak emission wavelength ranges of FAM, TAMRA,TET, and ROX.

In this embodiment, the set of filters preferably includes a 515 nmSchott Glass® filter 222A positioned in front of the first detector102A, a 550 nm Schott Glass® filter 222B positioned in front of thesecond detector 102B, a 570 nm Schott Glass® filter 222C positioned infront of the third detector 102C, and a 620 nm Schott Glass® filter 222Dpositioned in front of the fourth detector 102D. These Schott Glass®filters are commercially available from Schott Glass Technologies, Inc.of Duryea, Pa. The optics assembly 70 also includes a pair of 505 nmhigh pass filters 223 positioned in front of the first detector 102A, apair of 537 nm high pass filters 224 positioned in front of the seconddetector 102B, a pair of 565 nm high pass filters 225 positioned infront of the third detector 102C, and a pair of 605 nm high pass filters226 positioned in front of the fourth detector 102D.

Although it is presently preferred to position a pair of high passfilters in front of each detector for double filtering of light, asingle filter may be used in alternative embodiments. In addition, alens 242 is preferably positioned in each detection channel between thepair of high pass filters and the Schott Glass® filter for collimatingthe filtered light. The optics assembly 70 further includes a 605 nmhigh pass reflector 227, a mirror 228, a 565 nm low pass reflector 229,a 537 nm high pass reflector 230, and a 505 nm high pass reflector 231.The reflecting filters and mirrors 227–231 are preferably angularlyoffset by 30° from the high pass filters 223–226. As shown in FIG. 17,the detection assembly 70 also preferably includes a first aperture 238positioned between each detector and Schott Glass® filter 222 and anaperture 240 positioned between each lens 242 and Schott Glass® filter222. The apertures 238, 240 reduce the amount of stray or off-axis lightthat reaches the detectors 102A, 102B, 102C, and 102D.

Referring again to FIG. 16, the detection assembly 70 detects lightemitted from the chamber 17 in four emission wavelength ranges asfollows. The emitted light passes through the lens 232 and strikes the565 nm low pass reflector 229. The portion of the light having awavelength in the range of about 505 to 537 nm (corresponding to thepeak emission wavelength range of FAM) reflects from the 565 nm low passreflector 229, passes through the 537 nm high pass reflector 230,reflects from the 505 nm high pass reflector 231, passes through thepair of 505 nm high pass filters 223, through the lens 242, through the515 nm Schott Glass® filter 222A, and is detected by the first detector102A. Meanwhile, the portion of the light having a wavelength in therange of about 537 to 565 nm (corresponding to the peak emissionwavelength range of TET) reflects from the 565 nm low pass reflector229, reflects from the 537 nm high pass reflector 230, passes throughthe pair of 537 nm high pass filters 224, through the lens 242, throughthe 550 nm Schott Glass® filter 222B, and is detected by the seconddetector 102B.

Further, the portion of the light having a wavelength in the range ofabout 565 to 605 nm (corresponding to the peak emission wavelength rangeof TAMRA) passes through the 565 nm low pass reflector 229, through the605 nm high pass reflector 227, through the pair of 565 nm high passfilters 225, through the lens 242, through the 570 nm Schott Glass®filter 222C, and is detected by the third detector 102C. The portion ofthe light having a wavelength over 605 nm (corresponding to the peakemission wavelength range of ROX) passes through the 565 nm low passreflector 229, reflects from the 605 nm high pass reflector 227,reflects from the mirror 228, passes through the pair of 605 nm highpass filters 226, through the lens 242, through the 620 nm Schott Glass®filter 222D, and is detected by the fourth detector 102D. In operation,the outputs of detectors 102A, 102B, 102C, and 102D are analyzed todetermine the concentrations of each of the differentfluorescently-labeled analytes contained in the chamber 17, as will bedescribed in greater detail below.

FIG. 18 shows a multi-site reactor system 106 according to the presentinvention. The reactor system 106 comprises a thermal cycler 108 and acontroller 112, such as a personal or network computer. The thermalcycler 108 includes a base instrument 110 for receiving multipleheat-exchanging modules 60 (previously described with reference to FIG.6). The base instrument 110 has a main logic board with edge connectors114 for establishing electrical connections to the modules 60. The baseinstrument 110 also preferably includes a fan 116 for cooling itselectronic components. The base instrument 110 may be connected to thecontroller 112 using any suitable data connection, such as a universalserial bus (USB), ethernet connection, or serial line. It is presentlypreferred to use a USB that connects to the serial port of controller112. Alternatively, the controller may be built into the base instrument110.

The term “thermal cycling” is herein intended to mean at least onechange of temperature, i.e. increase or decrease of temperature, in asample. Therefore, samples undergoing thermal cycling may shift from onetemperature to another and then stabilize at that temperature,transition to a second temperature or return to the startingtemperature. The temperature cycle may be performed only once or may berepeated as many times as required to study or complete the particularchemical reaction of interest. Due to space limitations in patentdrawings, the thermal cycler 108 shown in FIG. 18 includes only sixteenreaction sites provided by the sixteen heat-exchanging modules 60arranged in two rows of eight modules each. It is to be understood,however, that the thermal cycler can include any number of desiredreaction sites, i.e., it can be configured as a multi-hundred siteinstrument for simultaneously processing hundreds of samples.Alternatively, it may be configured as a small, hand held,battery-operated instrument having, e.g., 1 to 4 reaction sites.

Each of the reaction sites in the thermal cycler 108 is provided by arespective one of the heat-exchanging modules 60. The modules 60 arepreferably independently controllable so that different chemicalreactions can be run simultaneously in the thermal cycler 108. Thethermal cycler 108 is preferably modular so that each heat-exchangingmodule 60 can be individually removed from the base instrument 110 forservicing, repair, or replacement. This modularity reduces downtimesince all the modules 60 are not off line to repair one, and theinstrument 110 can be upgraded and enlarged to add more modules asneeded. The modularity of the thermal cycler 108 also means thatindividual modules 60 can be precisely calibrated, and module-specificschedules or corrections can be included in the control programs, e.g.,as a series of module-specific calibration or adjustment charts.

In embodiments in which the base instrument 110 operates on externalpower, e.g. 110 V AC, the instrument preferably includes two powerconnections 122, 124. Power is received though the first connection 122and output through the second connection 124. Similarly, the instrument110 preferably includes network interface inlet and outlet ports 118,120 for receiving a data connection through inlet port 118 andoutputting data to another base instrument through outlet port 120. Asshown in the block diagram of FIG. 19, this arrangement permits multiplethermal cyclers 108A, 108B, 108C, 108D to be daisy-chained from onecontroller 112 and one external power source 128.

FIG. 20 is a schematic, block diagram of the base instrument 110. Thebase instrument includes a power supply 134 for supplying power to theinstrument and to each module 60. The power supply 134 may comprise anAC/DC converter for receiving power from an external source andconverting it to direct current, e.g., for receiving 110V AC andconverting it to 12V DC. Alternatively, the power supply 134 maycomprise a battery, e.g., a 12V battery. The base instrument 110 alsoincludes a microprocessor or microcontroller 130 containing firmware forcontrolling the operation of the base instrument 110 and modules 60. Themicrocontroller 130 communicates through a network interface 132 to thecontroller computer via a USB. Due to current limitations of processingpower, it is currently preferred to include at least one microcontrollerin the base instrument per sixteen modules 60. Thus if the baseinstrument has a thirty-two module capacity, at least twomicrocontrollers should be installed in the instrument 110 to controlthe modules.

The base instrument 110 further includes a heater power source andcontrol circuit 136, a power distributor 138, a data bus 140, and amodule selection control circuit 142. Due to space limitations in patentdrawings, control circuit 136, power distributor 138, data bus 140, andcontrol circuit 142 are shown only once in the block diagram of FIG. 20.However, the base instrument 110 actually contains one set of these fourfunctional components 136, 138, 140, 142 for each heat-exchanging module60. Thus, in the embodiment of FIG. 20, the base instrument 110 includessixteen control circuits 136, power distributors 138, data buses 140,and control circuits 142. Similarly, the base instrument 110 alsoincludes a different edge connector 131 for connecting to each of themodules 60, so that the instrument includes sixteen edge connectors forthe embodiment shown in FIG. 20. The edge connectors are preferably 120pin card edge connectors that provide cableless connection from the baseinstrument 110 to each of the modules 60. Each control circuit 136,power distributor 138, data bus 140, and control circuit 142 isconnected to a respective one of the edge connectors and to themicrocontroller 130.

Each heater power and source control circuit 136 is a power regulatorfor regulating the amount of power supplied to the heating element(s) ofa respective one of the modules 60. The source control circuit 136 ispreferably a DC/DC converter that receives a +12V input from the powersupply 134 and outputs a variable voltage between 0 and −24V. Thevoltage is varied in accordance with signals received from themicrocontroller 130. Each power distributor 138 provides −5 v, +5V,+12V, and GND to a respective module 60. The power distributor thussupplies power for the electronic components of the module. Each databus 140 provides parallel and serial connections between themicrocontroller 130 and the digital devices of a respective one of themodules 60. Each module selection controller 94 allows themicrocontroller 130 to address an individual module 60 in order to reador write control or status information.

FIG. 21 is a schematic, block diagram of the electronic components of aheat-exchanging module 60. Each module includes an edge connector 80 forcableless connection to a corresponding edge connector of the baseinstrument. The module also includes heater plates 50A, 50B each havinga resistive heating element as described above. The plates 50A, 50B arewired in parallel to receive power input 146 from the base instrument.The plates 50A, 50B also include temperature sensors 52, e.g.thermistors, that output analog temperature signals to ananalog-to-digital converter 154. The converter 154 converts the analogsignals to digital signals and routes them to the microcontroller in thebase instrument through the edge connector 80. The heat-exchangingmodule also includes a cooling system, such as a fan 66, for cooling theplates 50A, 50B. The fan 66 receives power from the base instrument andis activated by switching a power switch 164. The power switch 164 is inturn controlled by a control logic block 162 that receives controlsignals from the microcontroller in the base instrument.

The module further includes four light sources, such as LEDs 100, forexcitation of labeled analytes in the sample and four detectors 102,preferably photodiodes, for detecting fluorescent emissions from thesample. The module also includes an adjustable current source 150 forsupplying a variable amount of current (e.g., in the range of 0 to 30mA) to each LED to vary the brightness of the LED. A digital-to-analogconverter 152 is connected between the adjustable current source 150 andthe microcontroller of the base instrument to permit the microcontrollerto adjust the current source digitally. The adjustable current source150 may be used to ensure that each LED has about the same brightnesswhen activated. Due to manufacturing variances, many LEDs have differentbrightnesses when provided with the same amount of current. Thebrightness of each LED may be tested during manufacture of theheat-exchanging module and calibration data stored in a memory 160 ofthe module. The calibration data indicates the correct amount of currentto provide to each LED. The microcontroller reads the calibration datafrom the memory 160 and controls the current source 150 accordingly. Themicrocontroller may also control the current source 150 to adjust thebrightness of the LEDs 100 in response to optical feedback received fromthe detectors 102.

The module additionally includes a signal conditioning/gainselect/offset adjust block 156 comprised of amplifiers, switches,electronic filters, and a digital-to-analog converter. The block 156adjusts the signals from the detectors 102 to increase gain, offset, andreduce noise. The microcontroller in the base instrument controls block156 through a digital output register 158. The output register 158receives data from the microcontroller and outputs control voltages tothe block 156. The block 156 outputs the adjusted detector signals tothe microcontroller through the analog-to-digital converter 154 and theedge connector 80. The module also includes the memory 160, preferably aserial EEPROM, for storing data specific to the module, such ascalibration data for the LEDs 100, thermal plates 50A, 50B, andtemperature sensors 52, as well as calibration data for a deconvolutionalgorithm described in detail below.

Referring again to FIG. 18, the reactor system 106 may be configured formanual filling and pressurization of each reaction vessel 12 by a humanoperator. Manual use of the system is suitable for lower throughputembodiments. For higher throughput embodiments, however, the system 106preferably includes automated machinery, e.g., a pick-and-place machine,for filling and pressurizing each of the vessels 12.

FIG. 22 shows a schematic diagram of a pick-and-place machine 166 forautomatically filling and pressurizing a reaction vessel. The machine166 has a machine tip 168 for engaging a disposable pipette tip 170. Themachine tip 168 has an axial bore for communicating with the pipette tip170. The machine 166 also has controllable vacuum and pressure sourcesin communication with the machine tip 168 for aspirating and dispensingfluids using the pipette tip 170. The vacuum and pressure sources maycomprise, e.g., one or more syringe pumps, compressed air sources,pneumatic pumps, vacuum pumps, or connections to external sources ofpressure.

The machine tip may also be used to pick and place a reaction vessel orto insert a plug into the vessel, as is described below with referenceto FIGS. 23A–23D. The pick-and-place machine 166 also preferablyincludes an ejector plate 174 that slides down the machine tip 168 toeject the vessel, pipette tip, or plug from the machine tip. Referringagain to FIG. 2, the loading reservoir 38 preferably includes taperedwalls 40 for establishing a friction fit with the machine tip, therebyenabling the machine tip to pick and place the vessel 12. Similarly, theaspiration port 41 also preferably includes tapered walls 47 forestablishing a fit with the machine tip, so that the machine tip canpick and place the vessel 12 using either the loading reservoir 38 orthe aspiration port 41.

Referring again to FIG. 18, the controller 112 preferably includessoftware for controlling the thermal cycler 108 and the pick-and-placemachine to perform the functions described in the operation sectionbelow. These functions include providing a user interface to enable auser to select desired thermal processing parameters (e.g., set pointtemperatures and hold times at each temperature) and optical detectionparameters, automatic filling and pressurization of the vessels 12,thermal processing of the vessels according to the selected parameters,optical interrogation of the samples in the vessels, and recording ofthe optical data generated. The creation of software and/or firmware forperforming these functions can be performed by a computer programmerhaving ordinary skill in the art. Moreover, the software and/or firmwaremay reside solely in the controller 112 or may be distributed betweenthe controller and one or more microprocessors in the thermal cycler orpick-and-place machine. Alternatively, the controller 112 may simply bebuilt into the thermal cycler or pick-and-place machine.

In operation, the reactor system 106 is used to thermally process andoptically interrogate one or more samples. An exemplary use of thesystem 106 is for the amplification of nucleic acid in a sample (e.g.,using PCR) and for the optical detection of one or more target analytesin the sample. A user selects a desired thermal profile for the sampleusing, e.g., the keyboard or mouse of the controller 112. For example,for a PCR amplification, the user may select the thermal profile tobegin with a 30 second induction hold at 95° C., followed by 45 thermalcycles in which the sample is cycled between higher and lowertemperatures for denaturization, annealing, and polymerization. Forexample, each thermal cycle may include a first set point temperature of95° C. which is held for 1 second to denature double-stranded DNA,followed by a second set point temperature of 60° C. which is held for 6seconds for annealing of primers and polymerization.

FIGS. 23A–23D illustrate a preferred procedure for loading the sampleinto the vessel 12 and for sealing and pressurizing the vessel.Referring to FIG. 23A, an empty vessel 12 is first picked up by themachine tip 168 and placed into one of the heat-exchanging modules. Topick up the vessel 12, the machine tip 168 is inserted into theaspiration port 41 so that the tip 168 establishes a friction fit withthe tapered walls 47. Once the vessel 12 is inserted between the platesof a heat-exchanging module, the ejector plate 174 ejects the vesselfrom the machine tip 168.

As shown in FIG. 23B, the machine tip next picks up the pipette tip 170,and the sample is aspirated into the pipette tip. The sample is thendispensed into the loading reservoir 38 using the pipette tip 170. Thesample may be mixed with chemicals necessary for the intended reaction(e.g., PCR reagents and/or fluorescent probes) prior to being added tothe loading reservoir 38. Alternatively, the reagents may be added tothe loading reservoir 38 before or after the sample so that the reagentsand sample are mixed together in the reservoir 38. In anotherembodiment, the sample is introduced to the chemicals or reagents in thechamber 17. For example, the necessary reagents and/or fluorescentprobes for the intended reaction may be placed in the chamber 17 whenthe vessel is manufactured. The reagents are preferably placed in thechamber 17 in dried or lyophilized form so that they are adequatelypreserved until the vessel is used. When the fluid sample is added tothe chamber, it reconstitutes the dried or lyophilized reagents to formthe desired reaction mixture.

Referring to FIG. 23C, after the sample and/or reagents are dispensedinto the loading reservoir 38, the pipette tip 170 is inserted into theaspiration port 41. The pipette tip 170 establishes annular seal withthe tapered walls of the aspiration port 41 and applies vacuum pressureto the sample in the loading reservoir 38. The vacuum pressure isapplied to the sample through the channel 43, chamber 17, and channel39. In this manner, the pipette 170 draws (aspirates) the sample fromthe loading reservoir 38 into the chamber 17. Loading the sample intothe chamber 17 in this manner reduces the likelihood of any air bubblesforming in the chamber. Air bubbles would have a negative effect onsubsequent optical detection of target analytes in the sample. Followingloading of the sample into the chamber 17, the pipette tip 170 isejected.

As shown in FIG. 23D, the machine tip 168 then engages the cap 36 of theplug 22 and inserts the plug into the aperture 14, therebysimultaneously sealing the chamber 17, loading reservoir 38, andaspiration port 41 from the environment external to the vessel. The cap36 includes a tapered engagement aperture 46 for receiving andestablishing a friction fit with the machine tip 168. The machine tip168 also preferably includes an alignment pin 175 for aligning the plug22 in a desired angular orientation with respect to the aperture 14(i.e., so that the oval-shaped plug fits into the correspondinglyoval-shaped aperture). The alignment pin 175 provides a convenientmechanism for rotating the cap to the necessary angular orientationbefore inserting the plug 22 into the aperture 14. The cap 36 includesalignment apertures 48A, 48B, either one of which may receive thealignment pin 175. As the plug 22 is inserted, the sealing ring 32 sealsthe aperture 14 and the plug 22 compresses gas in the vessel to increasepressure in the chamber 17, preferably to about 8 to 15 psi aboveambient pressure, as previously discussed with reference to FIGS.24A–24B. When the plug 22 is inserted into the aperture 14, the catches35 engage the latches 33 to lock the plug 22 in place. After the plug 22is inserted, the ejector plate 174 ejects the plug 22 from the machinetip 168.

Although this embodiment of the pick-and-place machine is presentlypreferred, many other embodiments are possible. For example, the machinetip 168 may be designed to establish a vacuum fit with the cap 36.Alternatively, the pick-and-place machine may have a robotic gripper armfor gripping the plug 22 and inserting it into the aperture 14. Suitablepick-and-place machines for use in the system of the present inventionare commercially available as machines built to specification fromseveral suppliers, such as Tecan U.S. Inc. located at 4022 Stirrup CreekDrive, Durham, N.C. 27703.

Many modifications to the procedure described above for filling andpressurizing the vessel 12 are possible. For example, the vessel 12 maybe inserted between the plates of a respective heat-exchanging moduleafter the vessel is filled and pressurized rather than before. In thisembodiment, the vessel 12 is preferably held in a rack, tray, or similarsupport device during filling and pressurization. After the vessel 12 isfilled and capped, the machine tip 168 picks up the vessel 12 by the cap36 and inserts the chamber 17 of the vessel between the plates of aheat-exchanging module. The plug 22 is held in the aperture 14 duringthis movement by the latches 33 that engage the catches 35. After thevessel 12 is inserted, the ejector plate 174 ejects the cap 36 from themachine tip 168. The latches 33 are also effective for holding the cap36 on the vessel 12 as the vessel is removed from the module afterthermal processing and optical detection. Although automated filling andpressurization of the vessel 12 has been described herein, it is to beunderstood that the vessel may also be filled and pressurized manuallyby a human operator using, e.g., a pipette and human hands.

Referring again to FIG. 18, once a filled and pressurized reactionvessel 12 is placed between the thermal plates of a heat-exchangingmodule 60, the sample contained in the vessel is subjected to thethermal profile selected by the user. The controller 112 implementsproportional-integral-derivative (PID) control to execute the selectedthermal profile. Referring again to FIG. 21, the controller receivessignals indicating the temperatures of the plates 50A, 50B from thetemperature sensors 52. Polling of the plate temperatures preferablyoccurs every 100 milliseconds throughout the running of the temperatureprofile. After each polling, the controller averages the temperatures ofthe two plates 50A, 50B to determine an average plate temperature. Thecontroller then determines the difference (delta) between the profiletarget temperature, i.e. the set point temperature defined by the userfor the particular time in the profile, and the average platetemperature. Based on the relationship between the average platetemperature and the current target temperature, the controller controlsthe amount of power supplied to the heating elements on the plates 50A,50B or to the fan 66 as appropriate to reach or maintain the current setpoint temperature. Standard PID control is well known in the art andneed not be described further herein. The controller may optionallyimplement a modified version of PID control described in InternationalPublication Number WO 99/48608 published Sep. 30, 1999, the disclosureof which is incorporated by reference herein.

Referring again to FIGS. 14 and 16, the sample in the vessel 12 isoptically interrogated in real-time as the thermal profile is executedto determine if the sample contains one or more target analytes (e.g.,nucleic acid sequences of interest). The sample is preferably opticallyinterrogated once per thermal cycle at the lowest temperature in thecycle. Optical interrogation is preferably accomplished by sequentiallyactivating LEDs 100A, 100B, 100C, and 100D to excite differentfluorescently-labeled analytes in the sample and by detecting lightemitted (fluorescent output) from the chamber 17 using detectors 102A,102B, 102C, and 102D. Specific techniques for detecting analytes thatlabeled with dyes or probes are known in the art. Suitable examples ofoperation of the optical detection system of FIGS. 14–17 are given incommonly assigned U.S. Pat. No. 6,369,893 “Multi-Channel OpticalDetection System” the disclosure of which is incorporated by referenceherein.

In a typical implementation of the four-channel system, three of theoptical channels are used to detect target analytes (e.g., amplifiednucleic acid sequences) while the fourth channel is used to monitor aninternal control to check the performance of the system. For example,beta actin is often used as an internal control in nucleic acidamplification reactions because it has a predictable amplificationresponse and can be easily labeled and monitored to verify that theamplification is occurring properly.

One advantage of the system of the preferred embodiment is that itprovides extremely rapid heating and cooling of a sample. This rapidheating and cooling is particularly beneficial for nucleic acidamplification because of the increased speed with which theamplification may be accomplished and because it significantly reducesthe likelihood of creating unwanted and interfering side products, suchas PCR “primer-dimers” or anomalous amplicons. Another advantage of thesystem is that it provides for sensitive, real-time detection of one ormore analytes in a sample as the reaction is performed. Anotheradvantage is that the loading structure of the reaction vessel providesfor an extremely clean fill of the chamber without the formation of airbubbles that would be detrimental to optical detection. The loadingreservoir and aspiration port also eliminate the need to insert thepipette into the chamber 17. Consequently, the thickness of the chamberis not limited by the minimum practical pipette diameter which can beemployed in the sample transfer process. Thus, the chamber can have athickness less than the width or diameter of the aspiration device.

FIG. 30 shows another embodiment of the invention in which the plug 22includes first and second tongues 222A, 222B extending from the mainbody of the plug. The first tongue 222A is sized to be inserted into theinlet channel 39 that connects the loading reservoir 38 to the chamber17. Similarly, the second tongue 222B is sized to be inserted into theoutlet channel 43 that connects the aspiration port 41 to the chamber17. The tongues 222A, 222B are positioned with respect to the main bodyof the plug 22 such that when the plug 22 is inserted into the aperture14, the plugs 222A, 222B are inserted into the channels 39 and 43,respectively. The tongues 222A, 222B provide a physical obstacle forpreventing the reaction mixture R in the chamber 17 from refluxing (i.e.bubbling up or evaporating) into the loading reservoir 38 or aspirationport 41 as the mixture is heated. The tongues 222A, 222B thus preventsome vapor loss from the chamber 17 as the reaction mixture R isthermally processed. The tongues 222A, 222B should not form a seal withthe walls of the channels 39, 43. Such sealing would cause the chamber17 to become hydraulically locked, which is undesirable. Such ahydraulically locked condition could result in damage to the major walls18 or plates 50A, 50B (FIG. 4) and/or prevent the major walls 18 fromconforming to the surfaces of the plates 50A, 50B.

FIG. 25A shows a reaction vessel 180 according to another embodiment ofthe invention. Like the vessel of FIG. 2A, the vessel 180 has a rigidframe 186 defining the side walls of a reaction chamber 187, flexiblesheets attached to opposite sides of the frame 186 to form opposingmajor walls of the chamber, and a loading structure 188 extending fromthe frame 186 for loading a sample into the chamber. The loadingstructure 188 is preferably integrally molded with the frame 186. Theloading structure 188 defines a loading reservoir 190 connected to thechamber 187 by an inlet channel 194. The loading structure 188 alsodefines an aspiration port 192 connected to the chamber 187 by an outletchannel 196. The aspiration port 192 has tapered walls for establishinga seal with an aspiration device (e.g., a pipette tip), thereby enablingthe aspiration device to draw the sample from the loading reservoir 190into the chamber 187.

The vessel 180 differs from the vessel of FIG. 2A in the mechanism forsealing and pressurizing the vessel. The vessel 180 includes a cap 198having first and second plugs 200A, 200B for sealing the loadingreservoir 190 and the aspiration port 192, respectively, and forcompressing gas in the vessel, thereby increasing pressure in thechamber 187. The cap 198 may optionally include an engagement aperture46 for receiving and establishing a fit with a machine tip. The cap 198may also include alignment apertures 48A, 48B for receiving alignmentpins to permit automated picking and placing of the cap by apick-and-place machine.

The vessel 180 preferably includes flow control means for preventingsubstantial flow of the sample from the loading reservoir 190 to thereaction chamber 187 until the sample is drawn into the chamber 187 byan aspiration device. As previously described, suitable flow controlmeans include, but are not limited to: a channel having a narrow widthor diameter; one or more valves; one or more membranes; or one or morescreens. FIG. 25A shows one embodiment of the vessel in which the flowcontrol means is at least one portion of the channel 194 having asufficiently small width or diameter (e.g., 0.031 inches or less issuitable for most aqueous solutions having the viscosity of water) toprevent substantial flow of the sample (e.g., due to gravitationalforce) from the loading reservoir 190 to the chamber 187 until thesample is drawn into the chamber by application of a vacuum toaspiration port 192. FIG. 25B shows another embodiment of the vessel inwhich the flow control means is a valve 111 in the channel 194. Thereare many types of valves known in the art that are suitable for thispurpose including, e.g., a flap valve, check valve, or rotary valve.FIG. 25C shows another embodiment of the vessel in which the flowcontrol means is a membrane or screen 113 in the channel 194. Suitablemembranes include, e.g., burstable or piercable membranes that are burstor pierced to permit fluid flow through the channel 194. If a screen isused as the flow control component, the screen should have a pore sizesufficient to prevent substantial flow of the sample before a vacuum isapplied to the aspiration port 192 and permit flow of the sample whenthe vacuum is applied.

In operation, a sample is dispensed into the loading reservoir 190 usingan aspirating and dispensing device, e.g. a pipette. The pipette tip isthen inserted into the aspiration port 192 such that the pipette tipestablishes a seal with the tapered walls. The sample is then drawn(aspirated) from the loading reservoir 190 into the chamber 187 byapplication of vacuum pressure to the aspiration port 192. Followingloading of the sample into the chamber 187, the cap 198 is placed on thevessel 180 such that the plugs 200A, 200B are inserted into the loadingreservoir 190 and aspiration port 192, respectively. The plugs 200A,200B should be designed to seal the loading reservoir 190 and aspirationport 192, respectively, at exactly the same time. Otherwise, the samplemay be forced to squirt out of the loading reservoir 190 or theaspiration port 192. For this reason, the vessel described in FIGS. 1–2Ais presently preferred. The sealing aperture 14 ensures perfect sealingand pressurization of the vessel.

FIG. 26 shows an alternative embodiment of the invention in which thepressurization of the vessel 12 is performed by a pick-and-place machine282 having a machine head 284. The machine head 284 has an axial bore286 for communicating with the chamber 17 through the loading reservoir38 and aspiration port 41. The pick-and-place machine 282 also includesa regulated pressure source in fluid communication with the bore 286 forpressurizing the vessel 12 through the bore 286. The pressure source maycomprise, e.g., a syringe pump, compressed air source, pneumatic pump,or connection to an external air supply.

The system also preferably includes a disposable adapter 288 for placingthe bore 286 in fluid communication with the chamber 17. The adapter 288has an axial bore 290 that connects the bore 286 in the machine head tothe aperture 14 in the vessel. The adapter 288 is sized to be insertedinto the aperture 14 such that the adapter establishes a seal with thewalls of the aperture. The adapter 282 preferably comprises anelastomeric material, e.g., a thermalplastic elastomer (TPE) orsilicone. The adapter 288 preferably includes a one-way valve 292 (e.g.,a check valve) for preventing fluid from escaping from the vessel 12.

In operation, the vessel 12 is preferably placed into a heat-exchangingmodule and filled with a sample as previously described in the preferredembodiment. The vessel may be filled manually by a human operator, oralternatively, the pick-and-place machine 282 may include a pipette forfilling the vessel. After the chamber 17 is filled with the sample, themachine head 284 picks up the adapter 288 and inserts the adapter intothe aperture 14. To pick and place the adapter 288, the machine head 284preferably has a collet for gripping and releasing the adapter 288.Alternatively, the machine head may be sized to establish a press orfriction fit with the adapter 288. When inserted into the aperture 14,the adapter 288 establishes a seal with the walls of the aperture. Thepick-and-place machine 282 then transmits gas, preferably air, from thepressure source into the vessel 12 to increase the pressure in thechamber 17. The flow of air into the vessel 12 is stopped when thedesired pressurization of the chamber 17 is achieved.

The desired pressurization of the chamber 17 in this embodiment is thesame as that described in the preferred embodiment above. As shown inFIG. 4, the pressure in the chamber 17 should be sufficiently high toensure that the flexible major walls 18 of the chamber outwardly expandto contact and conform to the surfaces of the plates 50A, 50B. Thepressure should not be so great, however, that the walls 18 burst,become unattached from the frame 16, or deform the frame or plates. Itis presently preferred to pressurize the chamber 17 to a pressure in therange of 2 to 50 psi above ambient pressure. This range is preferredbecause 2 psi is generally enough pressure to ensure conformity betweenthe flexible walls 18 and the surfaces of the plates 50A, 50B, whilepressures above 50 psi may cause bursting of the walls 18 or deformationof the frame 16 or plates 50A, 50B. More preferably, the chamber 17 ispressurized to a pressure in the range of 8 to 15 psi above ambientpressure. This range is more preferred because it is safely within thepractical limits described above to allow for any manufacturing oroperational deviations from specification.

Referring again to FIG. 26, the machine head 284 is disengaged from theadapter 288 following the pressurization of the vessel 12. When themachine head 284 is disengaged from the adapter 288, the valve 292prevents fluid from escaping from the vessel 12. Thus, the chamber 17remains pressurized for thermal processing and the vessel 12 iseffectively sealed to prevent the sample in the vessel fromcontaminating the external environment. The remaining operation of thisembodiment is analogous to the operation of the preferred embodimentdescribed above.

FIG. 27 shows another embodiment of the invention in which thepressurization of vessel 12 is performed by a pick-and-place machine 300having a machine head 302 for manipulating a needle 306. The machinehead 302 has an axial bore 304 for communicating with the needle 306.The pick-and-place machine 300 has controllable vacuum and pressuresources in communication with the bore 304 for aspirating and dispensingfluids using the needle 306. The vacuum and pressure sources maycomprise, e.g., one or more syringe pumps, compressed air sources,pneumatic pumps, vacuum pumps, or connections to external sources ofpressure. The machine head 302 engages the needle 306 using any standardneedle fitting, such as a luer lock.

The system also includes an elastomeric plug 310 that is inserted intothe aperture 14 of the vessel such that the plug forms a seal with thewalls of the aperture. The needle 306 is inserted through the plug 310by the machine head 302 to pressurize the chamber 17. The elastomericplug 310 should be self-sealing so that it seals fluid within the vessel12 when the needle 306 is withdrawn from the plug 310. The plug 310 maybe inserted into the aperture 14 by a robotic arm or machine tip of thepick-and-place machine 300 or the plug may be manually inserted by ahuman operator.

In operation, the vessel 12 is preferably placed into a heat-exchangingmodule and filled with a sample as previously described in the preferredembodiment. The vessel may be filled manually by a human operator, oralternatively, the pick-and-place machine 300 may include a pipette forfilling the vessel. After the chamber 17 is filled with the sample, theself-sealing plug 310 is inserted into the aperture 14. The machine head302 then inserts the needle 306 through the plug 319 so that the tip ofthe needle is inside the vessel 12. The pick-and-place machine 300 thenflows gas, preferably air, from the controllable pressure source intothe vessel 12 through the needle 306 to increase pressure in the chamber17. The machine 300 then stops the flow of air when the desiredpressurization of the chamber 17 is achieved.

The desired pressurization of the chamber 17 in this embodiment is thesame as that described in previous embodiments, e.g., 5 to 50 psi andmore preferably 8 to 15 psi for the reasons discussed above. Followingpressurization, the machine head 302 retracts the needle 306 from theplug 310, and the plug 310 self seals to maintain the desired pressurein the vessel 12 for thermal processing. The remaining operation of thisembodiment is analogous to the operation of the preferred embodimentdescribed above.

FIG. 28 shows another embodiment of the invention in which the sealingand pressurization of vessel 12 is performed by a press 314 having aheated platen 316 for heat sealing a film or foil 318 to the portion ofthe loading structure 30 forming the seal aperture 14. The foil 318 ispreferably a laminate comprising a layer of metal (e.g., aluminum) ontop of a layer of polymeric material (e.g., polypropylene or polyester).In operation, the vessel 12 is preferably placed in a holder (e.g., atray or nest) that moves on an assembly line for automated filling,sealing, and pressurization of the vessel. In a first step, the chamber17 of the vessel is filled with a sample as previously described in thepreferred embodiment. After the chamber 17 is filled, the foil 318 isplaced on top of the loading structure 30 with the metal layer facingup. The foil 318 may be placed on the vessel manually by a humanoperator, or more preferably, by the robotic arm of a pick-and-placemachine. The vessel 12 is then moved under the heated platen 316 forsealing and pressurization. The platen 316 is then pressed to the top ofthe vessel 12 and the platen 316 heat seals the foil 318 to the vesselto seal the aperture 14.

As shown in FIG. 29, the heat from the platen 316 also melts the topportion of the loading structure 30, thereby collapsing an end of theaperture 14 to produce a collapsed zone 319. The volume capacity of thevessel 12 is therefore reduced. The reduction of the volume capacity ofthe vessel 12 after the port is sealed compresses air trapped in thevessel and causes the desired pressurization of the chamber 17. Thedesired pressurization of the chamber 17 in this embodiment is the sameas that described in the previous embodiments, e.g., 2 to 50 psi abovethe ambient pressure, and more preferably 8 to 15 psi above the ambientpressure. After the vessel 12 is sealed and pressurized in this manner,it is picked and placed into one of the heat-exchanging modules 60 (FIG.18) for thermal processing and optical detection. The remainingoperation of this embodiment is the same as the operation of thepreferred embodiment described above.

The desired pressurization of the chamber 17 may be achieved by use ofthe equation:P _(i) *V _(i) =P _(f) *V _(f),

where:

P_(i) is equal to the initial pressure in the vessel 12 prior to sealingthe aperture;

V_(i) is equal to the volume capacity of the vessel between the surfacelevel S and the top of the aperture 14 prior to sealing the vessel;

P_(f) is equal to the desired final pressure in the chamber 17; and

V_(f) is equal to the final volume capacity of the vessel between thesurface level S and the collapsed zone 319.

To ensure the desired final pressure P_(f) in the chamber 17, theheat-sealing of the vessel should reduce the volume capacity of thevessel such that the ratio of the volumes V_(i):V_(f) is substantiallyequal to the ratio of the pressures P_(f):P_(i). An engineer havingordinary skill in the art will be able to select suitable values for thevolumes V_(i) and V_(f) using the description and equation given above.For example, if the initial pressure P_(i) in the vessel is equal tostandard atmospheric pressure of about 14 psi, the desired finalpressure P_(f) is equal to 26 psi (the desired 12 psi above ambientpressure), and the initial volume capacity V_(i) is equal to 500 l, thenthe heat sealing of the vessel should reduce the volume capacity to avolume V_(f) of about 275 l. This is just one example of suitable valuesfor the initial and final volumes, and it is to be understood that thescope of the invention is not limited to this example. Many othersuitable values may be selected to achieve the desired ratios, as willbe apparent to one having ordinary skill in the art.

The various embodiments of the system of the present invention may finduse in many applications. The system may be utilized to perform chemicalreactions on samples, e.g., nucleic acid amplification, and to opticallydetect amplified target sequences. Although amplification by PCR hasbeen described herein, it will be appreciated by persons skilled in theart that the system may be utilized for a variety of otherpolynucleotide amplification reactions and ligand-binding assays. Suchadditional reactions may be thermally cycled or they may be carried outat a single temperature, e.g., isothermal nucleic acid amplification.Other applications of the system are intended to be within the scope ofthe invention where those applications require temperature control of asample and/or optical detection.

Summary, Ramifications, and Scope

Although the above description contains many specificities, these shouldnot be construed as limitations on the scope of the invention, butmerely as examples of some of the presently preferred embodiments. Manymodifications or substitutions may be made to the system and methodsdescribed without departing from the scope of the invention. Forexample, in one alternative embodiment, the reaction vessel has only oneflexible sheet forming a major wall of the reaction chamber. The rigidframe defines the other major wall of the chamber, as well as the sidewalls of the chamber. In this embodiment, the major wall formed by theframe should have a minimum thickness of about 0.05 inches (thepractical minimum thickness for injection molding), while the flexiblesheet may be as thin as 0.0005 inches. The advantage to this embodimentis that the manufacturing of the reaction vessel is simplified, andhence less expensive, since only one flexible sheet need be attached tothe frame. The disadvantage is that the heating and cooling rates of thesample are likely to be slower since the major wall formed by the framewill probably not permit as high a rate of heat transfer as the thin,flexible sheet.

In some embodiments, the system may have just one thermal surface forcontacting a flexible wall of the reaction vessel and one thermalelement for heating and/or cooling the thermal surface. The advantage tousing one thermal surface and one thermal element is that the system maybe manufactured less expensively. The disadvantage is that the heatingand cooling rates are likely to be about twice as slow. Further,although it is presently preferred that the thermal surfaces be formedby thermally conductive plates, each thermal surface may be provided byany rigid structure having a contact area for contacting a wall of thevessel. The thermal surface preferably comprises a material having ahigh thermal conductivity, such as ceramic or metal. Moreover, thethermal surface may comprise the surface of the thermal element itself.For example, the thermal surface may be the surface of an ultrasonictransducer that contacts the flexible wall of the chamber for ultrasonicheating and/or lysing of the sample in the chamber. Alternatively, thethermal surface may be the surface of a thermoelectric device thatcontacts the wall to heat and/or cool the chamber. In addition, thevessel may have a heated lid or cap.

The filters used in the optics assemblies may be designed to provideexcitation and emission light in any wavelength ranges of interest, notjust the specific wavelength ranges described above. The choice offluorescent dyes for any given application depends upon the analytes ofinterest. One skilled in the art will realize that differentcombinations of light sources, filters, or filter wavelengths may beused to accommodate the different peak excitation and emission spectraof the selected dyes. Moreover, although blue and green light sourcesare presently preferred, different color light sources, such asblue-green, red, or amber LEDs, may be used in the system. Further,infrared or ultraviolet light sources may be used.

Moreover, although fluorescence excitation and emission detection is apreferred embodiment, optical detection methods such as those used inabsorption and/or transmission with on-axis geometries may also beapplied to the system of the present invention. Alternative geometries,such as on-axis alignments of light sources and detectors, can be usedto monitor changes in dye concentrations and physical conditions(temperature, pH, etc.) of a reaction by measuring absorption of theillumination. The optics may also be used to measure time decayfluorescence. Additionally, the optics are not limited to detectionbased upon fluorescent labels. The optics system may be applicable todetection based upon phosphorescent labels, chemiluminescent labels, orelectrochemiluminescent labels.

The scope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims along with their full scope ofequivalents.

1. A system for controlling the temperature of a sample, the systemcomprising: a) a reaction vessel having: i) a reaction chamber definedby two opposing major walls and side walls connecting the major walls toeach other, at least one of the major walls comprising a sheet or film;ii) a loading reservoir for receiving the sample prior to loading thesample into the reaction chamber, the loading reservoir being connectedto the reaction chamber via a first channel; and iii) an aspiration portconnected to the reaction chamber via a second channel; b) an aspirationdevice for establishing a seal with the aspiration port and for drawingthe sample from the loading reservoir into the reaction chamber, whereinthe vessel further includes flow control means for preventingsubstantial flow of the sample from the loading reservoir into thereaction chamber until the sample is drawn into the chamber by theaspiration device; c) at least one heating or cooling surface forcontacting the sheet or film, the sheet or film being sufficientlyflexible to conform to the surface; and d) at least one thermal elementfor heating or cooling the surface to induce a temperature change in thereaction chamber.
 2. The system of claim 1, wherein the flow controlmeans comprises at least one portion of the first channel having asufficiently small width or diameter to prevent substantial flow of thesample from the loading reservoir to the chamber until the sample isdrawn into the chamber by the aspiration device.
 3. The system of claim1, wherein the flow control means comprises at least one valve in thefirst channel.
 4. The system of claim 1, wherein the flow control meanscomprises at least one membrane or screen in the first channel.
 5. Thesystem of claim 1, wherein the system includes at least two heating orcooling surfaces positioned to receive the vessel between them such thatthe surfaces contact the major walls, and wherein each of the majorwalls comprises a flexible sheet or film.
 6. The system of claim 5,wherein the heating or cooling surfaces are the surfaces of opposingplates positioned to receive the vessel between them, each of the plateshaving a thermal mass less than 1 J/° C.
 7. The system of claim 1,wherein each of the plates comprises a ceramic material, and wherein theat least one thermal element comprises heating elements coupled to theplates.
 8. The system of claim 1, wherein at least two of the side wallsof the chamber are optically transmissive, and wherein the systemfurther includes optics for optically interrogating the chamber whilethe sheet or film is in contact with the heating or cooling surface, theoptics comprising at least one light source for transmitting light tothe chamber through a first one of the optically transmissive walls andat least one detector for detecting light exiting the chamber through asecond one of the optically transmissive walls.
 9. The system of claim8, wherein the optically transmissive walls are angularly offset fromeach other by about 90°.
 10. The system of claim 9, further comprisingat least one controller for controlling the operation of the thermalelement, light source, and detector.
 11. The system of claim 1, whereinthe chamber has a thickness less than the width or diameter of theaspiration device.
 12. The system of claim 1, wherein the ratio of thewidth of the chamber to the thickness of the chamber is at least 4:1,and wherein the thickness of the chamber is less than or equal to 3 mm.13. The system of claim 1, further comprising means for increasing thepressure in the chamber, wherein the pressure increase in the chamber issufficient to force the sheet or film to conform to the surface.
 14. Thesystem of claim 13, wherein the vessel further includes a seal apertureextending over an outer end of the loading reservoir and an outer end ofthe aspiration port, and wherein the means for increasing the pressurein the chamber comprises a plug that is insertable into the aperture tocompress gas in the vessel.
 15. The system of claim 14, wherein thevessel includes an inner surface defining the seal aperture, and whereinthe inner surface has at least one pressure control groove formedtherein, the pressure control groove extending to a predetermined depthin the aperture to allow gas to escape from the aperture until the plugreaches the predetermined depth.
 16. The system of claim 15, furthercomprising an automated machine for inserting the plug into theaperture, wherein the machine has a machine tip for engaging the plug,and wherein the plug includes a cap having an engagement aperture forreceiving and establishing a fit with the machine tip.
 17. The system ofclaim 14, wherein the plug includes a cap having latches, and whereinthe vessel further comprises catches for engaging the latches, therebysecuring the plug in the aperture.
 18. The system of claim 13, whereinthe means for increasing the pressure in the chamber comprises first andsecond plugs which are inserted into the loading reservoir and theaspiration port, respectively, to compress gas in the vessel andincrease pressure in the chamber.
 19. The system of claim 13, whereinthe means for increasing pressure in the chamber comprises: i) a machinehead for communicating with the vessel; and ii) a pressure source forpressurizing the chamber through the machine head.
 20. The system ofclaim 19, wherein the vessel further includes a seal aperture extendingover an outer end of the loading reservoir and an outer end of theaspiration port, the system further comprises an adapter for placing themachine head in fluid communication with the chamber, and the adapter issized to be inserted into the aperture.
 21. The system of claim 20,wherein the adapter includes a valve for preventing fluid from escapingfrom the vessel.
 22. The system of claim 13, wherein the vessel furtherincludes a seal aperture extending over an outer end of the loadingreservoir and an outer end of the aspiration port, and wherein the meansfor increasing pressure in the chamber comprises: i) an elastomeric plugfor sealing the aperture; and ii) a needle for injecting fluid into thevessel through the plug.
 23. The system of claim 13, wherein the vesselfurther includes a seal aperture extending over an outer end of theloading reservoir and an outer end of the aspiration port, and whereinthe means for increasing pressure in the chamber comprises a platen forheat sealing a film or foil to the vessel to seal the aperture.
 24. Thesystem of claim 1, further comprising a pick-and-place machine having amachine tip for engaging the vessel, wherein either the loadingreservoir or the aspiration port has tapered walls for establishing afit with the machine tip, thereby enabling the machine tip to pick andplace the vessel.
 25. A system for controlling the temperature of asample, the system comprising: a) a reaction vessel having: i) areaction chamber; ii) a loading reservoir for receiving the sample,wherein the loading reservoir is connected to the reaction chamber via afirst channel; iii) an aspiration port connected to the reaction chambervia a second channel; and iv) a seal aperture extending over an outerend of the loading reservoir and an outer end of the aspiration port; b)an aspiration device for establishing a seal with the aspiration portand for drawing the sample from the loading reservoir into the reactionchamber, wherein the vessel further includes flow control means forpreventing substantial flow of the sample from the loading reservoirinto the reaction chamber until the sample is drawn into the reactionchamber by the aspiration device; c) a plug that is insertable into theaperture; and d) at least one thermal element for heating or cooling thereaction chamber.
 26. The system of claim 25, wherein the flow controlmeans comprises at least one portion of the first channel having asufficiently small width or diameter to prevent substantial flow of thesample from the loading reservoir to the chamber until the sample isdrawn into the chamber by the aspiration device.
 27. The system of claim25, wherein the flow control means comprises at least one valve in thefirst channel.
 28. The system of claim 25, wherein the flow controlmeans comprises at least one membrane or screen in the first channel.29. The system of claim 25, wherein the reaction chamber is defined bytwo opposing major walls and side walls connecting the major walls toeach other, at least one of the major walls comprises a sheet or film,and the at least one thermal element comprises: i) at least one heatingor cooling surface for contacting the sheet or film, the sheet or filmbeing sufficiently flexible to conform to the surface; and ii) means forheating or cooling the surface.
 30. The system of claim 25, wherein thevessel includes a rigid frame defining side walls of the chamber, thevessel includes two sheets or films attached to opposite sides of theframe to form two opposing major walls of the chamber, and the at leastone thermal element comprises: i) opposing plates positioned to receivethe vessel between them such that the plates contact the major walls;and ii) means for heating or cooling the plates.
 31. The system of claim30, wherein each of the plates comprises a ceramic material, and whereineach of the plates has a thickness less than or equal to 1 mm.
 32. Thesystem of claim 30, wherein the means for heating or cooling the platescomprises resistive heating elements coupled to the plates.
 33. Thesystem of claim 30, wherein each of the plates has a thermal mass lessthan or equal to 1 J/° C.
 34. The system of claim 25, wherein thereaction chamber is defined by a plurality of walls, at least two of thewalls are optically transmissive, and the system further comprisesoptics for optically interrogating the chamber, the optics comprising atleast one light source for transmitting light to the chamber through afirst one of the optically transmissive walls and at least one detectorfor detecting light exiting the chamber through a second one of theoptically transmissive walls.
 35. The system of claim 34, wherein theoptically transmissive walls are angularly offset from each other byabout 90°.
 36. The system of claim 34, further comprising at least onecontroller for controlling the operation of the thermal element, lightsource, and detector.
 37. The system of claim 25, wherein the reactionchamber has a thickness less than the width or diameter of theaspiration device.
 38. The system of claim 25, wherein the ratio of thewidth the reaction chamber to the thickness of the reaction chamber isat least 4:1, and wherein the thickness of the reaction chamber is lessthan or equal to 3 mm.
 39. The system of claim 25, wherein the plug issized to compress gas in the vessel, thereby increasing pressure in thereaction chamber to at least 2 pounds per square inch above ambientpressure.
 40. The system of claim 25, further comprising an automatedmachine for inserting the plug into the aperture, wherein the machinehas a machine tip for engaging the plug, and wherein the plug includes acap having an engagement aperture for receiving the machine tip.
 41. Thesystem of claim 25, wherein the plug includes a cap having latches, andwherein the vessel further comprises catches for engaging the latches,thereby securing the plug in the aperture.
 42. A system for loading asample into a reaction vessel and for controlling the temperature of thesample in the vessel, wherein the vessel includes a reaction chamber, aloading reservoir connected to the reaction chamber via a first channel,an aspiration port connected to the reaction chamber via a secondchannel, and a seal aperture extending over an outer end of the loadingreservoir and an outer end of the aspiration port, the systemcomprising: a) an aspiration and dispensing device for dispensing thesample into the loading reservoir, for establishing a seal with theaspiration port, and for drawing the sample from the loading reservoirinto the reaction chamber, wherein the vessel includes flow controlmeans for preventing substantial flow of the sample from the loadingreservoir into the reaction chamber until the sample is drawn into thereaction chamber by the aspiration and dispensing device; b) anautomated machine for inserting a plug into the seal aperture afterloading the sample into the chamber; and c) at least one thermal elementfor heating or cooling the reaction chamber.
 43. The system of claim 42,wherein the flow control means comprises at least one portion of thefirst channel having a sufficiently small width or diameter to preventsubstantial flow of the sample from the loading reservoir to the chamberuntil the sample is drawn into the chamber by the aspiration device. 44.The system of claim 42, wherein the flow control means comprises atleast one valve in the first channel.
 45. The system of claim 42,wherein the flow control means comprises at least one membrane or screenin the first channel.
 46. The system of claim 42, wherein the at leastone thermal element comprises: i) opposing plates positioned to receivethe vessel between them such that the plates contact major walls of thereaction chamber; and ii) means for heating or cooling the plates. 47.The system of claim 46, wherein each of the plates comprises a ceramicmaterial, and wherein each of the plates has a thickness less than orequal to 1 mm.
 48. The system of claim 46, wherein the means for heatingor cooling the plates comprises resistive heating elements coupled tothe plates.
 49. The system of claim 46, wherein each of the plates has athermal mass less than or equal to 1 J/° C.
 50. The system of claim 42,wherein the reaction chamber is defined by a plurality of walls, atleast two of the walls are optically transmissive, and the systemfurther comprises optics for optically interrogating the chamber, theoptics comprising at least one light source for transmitting light tothe chamber through a first one of the optically transmissive walls andat least one detector for detecting light exiting the chamber through asecond one of the optically transmissive walls.
 51. The system of claim50, further comprising at least one controller for controlling theoperation of the thermal element, light source, and detector.
 52. Areaction vessel comprising: a) a reaction chamber defined by twoopposing major walls and side walls connecting the major walls to eachother, wherein at least one of the major walls comprises a sheet or filmsufficiently flexible to conform to a heating or cooling surface, andwherein at least two of the side walls are optically transmissive toprovide optical windows to the reaction chamber; b) a loading reservoirfor receiving the sample prior to loading the sample into the reactionchamber, wherein the loading reservoir is connected to the reactionchamber via a first channel; and c) an aspiration port for establishinga seal with an aspiration device, the aspiration port being connected tothe reaction chamber via a second channel, thereby enabling theaspiration device to draw the sample from the loading reservoir into thechamber, wherein the vessel further includes flow control means forpreventing substantial flow of the sample from the loading reservoirinto the reaction chamber until the sample is drawn into the reactionchamber by the aspiration device.
 53. The vessel of claim 52, whereinthe flow control means comprises at least one portion of the firstchannel having a sufficiently small width or diameter to preventsubstantial flow of the sample from the loading reservoir to thereaction chamber until the sample is drawn into the reaction chamber bythe aspiration device.
 54. The vessel of claim 52, wherein the flowcontrol means comprises at least one valve in the first channel.
 55. Thevessel of claim 52, wherein the flow control means comprises at leastone membrane or screen in the first channel.
 56. The vessel of claim 52,wherein the vessel includes a rigid frame defining the side walls, eachof the two opposing major walls comprises a flexible sheet or filmattached to the frame, and the vessel includes a loading structureextending from the frame, the loading structure defining the loadingreservoir and the aspiration port.
 57. The vessel of claim 52, whereinthe optically transmissive walls are angularly offset from each other byabout 90°.
 58. The vessel of claim 52, wherein the ratio of the widththe reaction chamber to the thickness of the reaction chamber is atleast 4:1, and wherein the thickness of the reaction chamber is lessthan or equal to 3 mm.
 59. The vessel of claim 52, further comprising:i) a seal aperture extending over an outer end of the loading reservoirand an outer end of the aspiration port; and ii) a plug that isinsertable into the aperture.
 60. The vessel of claim 59, wherein theplug is sized to compress gas in the vessel, thereby increasing pressurein the reaction chamber.
 61. The vessel of claim 59, wherein the vesselincludes an inner surface defining the seal aperture, and wherein theinner surface has at least one pressure control groove formed therein,the pressure control groove extending to a predetermined depth in theaperture to allow gas to escape from the aperture until the plug reachesthe predetermined depth.
 62. The vessel of claim 52, wherein the plugincludes a cap having an engagement aperture for receiving andestablishing a fit with a machine tip.
 63. The vessel of claim 52,wherein the plug includes a cap having latches, and wherein the vesselfurther comprises catches for engaging the latches, thereby securing theplug in the aperture.
 64. The vessel of claim 52, wherein either theloading reservoir or the aspiration port has tapered walls forestablishing a fit with a machine tip, thereby enabling the machine tipto pick and place the vessel.
 65. The vessel of claim 52, furthercomprising dried or lyophilized reagents in the reaction chamber.
 66. Areaction vessel comprising: a) a reaction chamber; b) a loadingreservoir for receiving the sample prior to loading the sample into thereaction chamber, the loading reservoir being connected to the chambervia a first channel; c) an aspiration port for establishing a seal withan aspiration device, the aspiration port being connected to thereaction chamber via a second channel thereby enabling the aspirationdevice to draw the sample from the loading reservoir into the reactionchamber, wherein the vessel further includes flow control means forpreventing substantial flow of the sample from the loading reservoirinto the reaction chamber until the sample is drawn into the reactionchamber by the aspiration device; and d) a seal aperture, extending overan outer end of the loading reservoir and an outer end of the aspirationport, for receiving a plug that is inserted into the aperture afterloading the sample into the reaction chamber.
 67. The vessel of claim66, wherein the flow control means comprises at least one portion of thefirst channel having a sufficiently small width or diameter to preventsubstantial flow of the sample from the loading reservoir to thereaction chamber until the sample is drawn into the reaction chamber bythe aspiration device.
 68. The vessel of claim 66, wherein the flowcontrol means comprises at least one valve in the first channel.
 69. Thevessel of claim 66, wherein the flow control means comprises at leastone membrane or screen in the first channel.
 70. The vessel of claim 66,wherein the reaction chamber is defined by two opposing major walls andside walls connecting the major walls to each other, and wherein atleast one of the major walls comprises a sheet or film sufficientlyflexible to conform to a heating or cooling surface.
 71. The vessel ofclaim 70, wherein at least two of the side walls are opticallytransmissive to provide optical windows to the reaction chamber.
 72. Thevessel of claim 70, wherein the vessel includes a rigid frame definingthe side walls, each of the two opposing major walls comprises aflexible sheet or film attached to the frame, and the vessel includes aloading structure extending from the frame, the loading structuredefining the loading reservoir and the aspiration port.
 73. The vesselof claim 71, wherein the optically transmissive walls are angularlyoffset from each other by about 90°.
 74. The vessel of claim 66, whereinthe ratio of the width the reaction chamber to the thickness of thereaction chamber is at least 4:1, and wherein the thickness of thereaction chamber is less than or equal to 3 mm.
 75. The vessel of claim66, wherein the plug is sized to compress gas in the vessel, therebyincreasing pressure in the reaction chamber.
 76. The vessel of claim 66,wherein the pressure increase is at least 2 pounds per square inch. 77.The vessel of claim 66, wherein the pressure increase is in the range of8 to 15 pounds per square inch.
 78. The vessel of claim 66, wherein thevessel includes an inner surface defining the seal aperture, and whereinthe inner surface has at least one pressure control groove formedtherein, the pressure control groove extending to a predetermined depthin the aperture to allow gas to escape from the aperture until the plugreaches the predetermined depth.
 79. The vessel of claim 66, wherein theplug includes a cap having an engagement aperture for receiving andestablishing a fit with a machine tip.
 80. The vessel of claim 66,wherein the plug includes a cap having latches, and wherein the vesselfurther comprises catches for engaging the latches, thereby securing theplug in the aperture.
 81. The vessel of claim 66, wherein either theloading reservoir or the aspiration port has tapered walls forestablishing a fit with a machine tip, thereby enabling the machine tipto pick and place the vessel.
 82. The vessel of claim 66, furthercomprising dried or lyophilized reagents in the reaction chamber.